Virus Causing Respiratory Tract Illness in Susceptible Mammals

ABSTRACT

The invention relates to the field of virology. The invention provides an isolated essentially mammalian negative-sense single-stranded RNA virus (MPV) within the subfamily Pneumovirinae of the family Paramyxoviridae and identifiable as phylogenetically corresponding to the genus  Metapneumovirus  and components thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/466,811, with a filing date of Mar. 4, 2004, as national stageapplication of International Application PCT/NL02/00040, internationalfiling date Jan. 18, 2002, which claimed priority to European PatentApplication Serial No. 01200213.5, filed Jan. 19, 2001, and to EuropeanPatent Application Serial No. 01203985.5, filed Oct. 18, 2001, thedisclosure of each of which is hereby incorporated herein in itsentirety by this reference.

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Pursuant to 37 C.F.R. §1.821(c) or (e), a file containing a PDF versionof the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.The transmittal documents of this application include a Request toTransfer CRF from the parent application.

TECHNICAL FIELD

The invention relates to the field of virology.

BACKGROUND

In the past decades several etiological agents of mammalian disease, inparticular of respiratory tract illnesses (RTI), in particular ofhumans, have been identified. Classical etiological agents of RTI withmammals are respiratory syncytial viruses belonging to the genusPneumovirus found with humans (hRSV) and ruminants such as cattle orsheep (bRSV and/or oRSV). In human RSV differences in reciprocalcross-neutralization assays, reactivity of the G proteins inimmunological assays and nucleotide sequences of the G gene are used todefine 2 hRSV antigenic subgroups. Within the subgroups the aa sequencesshow 94% (subgroup A) or 98% (subgroup B) identity, while only 53% aasequence identity is found between the subgroups. Additional variabilityis observed within subgroups based on monoclonal antibodies, RT-PCRassays and RNAse protection assays. Viruses from both subgroups have aworldwide distribution and may occur during a single season. Infectionmay occur in presence of pre-existing immunity and the antigenicvariation is not strictly required to allow re-infection. See, forexample, W. M. Sullender, Respiratory Syncytial Virus Genetic andAntigenic Diversity, Clinical Microbiology Reviews, 2000, 13(1):1-15; P.L. Collins, K. McIntosh, and R. M. Chanock, Respiratory syncytial virus,Fields virology, ed. B. N. Knipe, P. M. Howley, 1996, Philadelphia:Lippencott-Raven, pp. 1313-1351; P. R. Johnson, et al., The Gglycoprotein of human respiratory syncytial viruses of subgroups A andB: extensive sequence divergence between antigenically related proteins,Proc. Natl. Acad. Sci. U.S.A., 1987, 84(16):5625-9; P. L. Collins, Themolecular Biology of Human Respiratory Syncytial Virus (RSV) of theGenus Pneumovirus, in The Paramyxoviruses, D. W. Kingsbury, Editor,1991, Plenum Press: New York. p. 103-153.

Another classical Pneumovirus is the pneumonia virus of mice (PVM), ingeneral only found with laboratory mice. However, a proportion of theillnesses observed among mammals can still not be attributed to knownpathogens.

The invention provides an isolated essentially mammalian negative-sensesingle-stranded RNA virus (MPV) belonging to the sub-familyPneumovirinae of the family Paramyxoviridae and identifiable asphylogenetically corresponding to the genus Metapneumovirus. The virusis identifiable as phylogenetically corresponding to the genusMetapneumovirus by determining a nucleic acid sequence of the virus andtesting it in phylogenetic analyses, for example wherein maximumlikelihood trees are generated using 100 bootstraps and 3 jumbles andfinding it to be more closely phylogenetically corresponding to a virusisolate deposited as I-2614 with CNOM, Paris than it is corresponding toa essentially avian virus isolate of avian Pneumovirus (APV) also knownas turkey rhinotracheitis virus (TRTV), the etiological agent of avianrhinotracheitis. For phylogenetic analyses, it is most useful to obtainthe nucleic acid sequence of a non-MPV as outgroup to be compared with,a very useful outgroup isolate can be obtained from avian Pneumovirusserotype C (APV-C), as is for example demonstrated in FIG. 5 herein.

Although phylogenetic analyses provides a convenient method ofidentifying a virus as an MPV several other possibly morestraightforward albeit somewhat more course methods for identifying thevirus or viral proteins or nucleic acids from the virus are herein alsoprovided. As a rule of thumb an MPV can be identified by the percentagesof a homology of the virus, proteins or nucleic acids to be identifiedin comparison with isolates, viral proteins, or nucleic acids identifiedherein by sequence or deposit. It is generally known that virus species,especially RNA virus species, often constitute a quasi species wherein acluster of the viruses displays heterogeneity among its members. Thus itis expected that each isolate may have a somewhat different percentagerelationship with one of the various isolates as provided herein.

When one wishes to compare with the deposited virus I-2614, theinvention provides an isolated essentially mammalian negative-sensesingle-stranded RNA virus (MPV) belonging to the sub-familyPneumovirinae of the family Paramyxoviridae and identifiable asphylogenetically corresponding to the genus Metapneumovirus bydetermining an amino acid sequence of the virus and determining that theamino acid sequence has a percentage amino acid homology to a virusisolate deposited as I-2614 with CNCMK Paris which is essentially higherthan the percentages provided herein for the L protein, the M protein,the N protein, the P protein, or the F protein, in comparison with APV-Cor, likewise, an isolated essentially mammalian negative-sensesingle-stranded RNA virus (NPV) belonging to the sub-familyPneumovirinae of the family Paramyxoviridae is provided as identifiableas phylogenetically corresponding to the genus Metapneumovirus bydetermining a nucleic acid sequence of the virus and determining thatthe nucleic acid sequence has a percentage nucleic acid identity to avirus isolate deposited as I-2614 with CNCM, Paris which is essentiallyhigher than the percentages identified herein for the nucleic acidsencoding the L protein, the M protein, the N protein, the P protein, orthe F protein as identified herein below in comparison with APV-C.

Again as a rule of thumb one may consider an MPV as belonging to one ofthe two serological groups of MPV as identified herein when the isolatesor the viral proteins or nuclear acids of the isolates that need to beidentified have percentages homology that fall within the bounds andmetes of the percentages of homology identified herein for both separategroups, taking isolates 00-1 or 99-1 as the respective isolates ofcomparison. However, when the percentages of homology are smaller orthere is more need to distinguish the viral isolates from for exampleAPV-C it is better advised to resort to the phylogenetic analyses asidentified herein.

Again one should keep in mind that the percentages can vary somewhatwhen other isolates are selected in the determination of the percentageof homology.

With the provision of this MPV, the invention provides diagnostic meansand methods and therapeutic means and methods to be employed in thediagnosis and/or treatment of disease, in particular of respiratorydisease, in particular of mammals, more in particular in humans.However, due to the, albeit distant, genetic relationship of theessentially mammalian MPV with the essentially avian APV, in particularwith APV-C, the invention also provides means and methods to be employedin the diagnosis and treatment of avian disease. In virology, it is mostadvisory that diagnosis and/or treatment of a specific viral infectionis performed with reagents that are most specific for the specific viruscausing the infection. In this case this means that it is preferred thatthe diagnosis and/or treatment of an MPV infection is performed withreagents that are most specific for MPV. This by no means howeverexcludes the possibility that less specific, but sufficientlycross-reactive reagents are used instead, for example because they aremore easily available and sufficiently address the task at hand. Hereinit is for example provided to perform virological and/or serologicaldiagnosis of MPV infections in mammals with reagents derived from APV,in particular with reagents derived from APV-C, in the detaileddescription herein it is for example shown that sufficiently trustworthyserological diagnosis of MPV infections in mammals can be achieved byusing an ELISA specifically designed to detect APV antibodies in birds.A particular useful test for this purpose is an ELISA test designed forthe detection of APV antibodies (e.g., in serum or egg yolk), onecommercially available version of which is known as APV-Ab SVANOVIR®which is manufactured by SVANOVA Biotech AB, Uppsal Science Park GluntenSE-751 83 Uppsala Sweden. The reverse situation is also the case, hereinit is for example provided to perform virological and/or serologicaldiagnosis of APV infections in mammals with reagents derived from MPV,in the detailed description herein it is for example shown thatsufficiently trustworthy serological diagnosis of APV infections inbirds can be achieved by using an ELISA designed to detect MPVantibodies. Considering that antigens and antibodies have a lock-and-keyrelationship, detection of the various antigens can be achieved byselecting the appropriate antibody having sufficient cross-reactivity.Of course, for relying on such cross-reactivity, it is best to selectthe reagents (such as antigens or antibodies) under guidance of theamino acid homologies that exist between the various (glyco)proteins ofthe various viruses, whereby reagents relating to the most homologousproteins will be most useful to be used in tests relying on thecross-reactivity.

For nucleic acid detection, it is even more straightforward, instead ofdesigning primers or probes based on heterologous nucleic acid sequencesof the various viruses and thus that detect differences between theessentially mammalian or avian Metapneumoviruses, it suffices to designor select primers or probes based on those stretches of virus-specificnucleic acid sequences that show high homology. In general, for nucleicacid sequences, homology percentages of 90% or higher guaranteesufficient cross-reactivity to be relied upon in diagnostic testsutilizing stringent conditions of hybridization.

The invention for example provides a method for virologically diagnosinga MPV infection of an animal in particular of a mammal, more inparticular of a human being, comprising determining in a sample of theanimal the presence of a viral isolate or component thereof by reactingthe sample with a MPV specific nucleic acid a or antibody according tothe invention, and a method for serologically diagnosing an MPVinfection of a mammal comprising determining in a sample of the mammalthe presence of an antibody specifically directed against an MPV orcomponent thereof by reacting the sample with a MPV-specificproteinaceous molecule or fragment thereof or an antigen according tothe invention. The invention also provides a diagnostic kit fordiagnosing an MPV infection comprising an MPV, an MPV-specific nucleicacid, proteinaceous molecule or fragment thereof, antigen and/or anantibody according to the invention, and preferably a means fordetecting the MPV, MPV-specific nucleic acid, proteinaceous molecule orfragment thereof, antigen and/or an antibody, the means for examplecomprising an excitable group such as a fluorophore or enzymaticdetection system used in the art (examples of suitable diagnostic kitformat comprise IF, ELISA, neutralization assay, RT-PCR assay). Todetermine whether an as yet unidentified virus component or syntheticanalogue thereof such as nucleic acid, proteinaceous molecule orfragment thereof can be identified as MPV-specific, it suffices toanalyze the nucleic acid or amino acid sequence of the component, forexample for a stretch of the nucleic acid or amino acid, preferably ofat least 10, more preferably at least 25, more preferably at least 40nucleotides or amino acids (respectively), by sequence homologycomparison with known MPV sequences and with known non-MPV sequencesAPV-C is preferably used) using for example phylogenetic analyses as.provided herein. Depending on the degree of relationship with the MPV ornon-MPV sequences, the component or synthetic analogue can beidentified.

The invention also provides method for virologically diagnosing an MPVinfection of a mammal comprising determining in a sample of the mammalthe presence of a viral isolate or component thereof by reacting thesample with a cross-reactive nucleic acid derived from APV (preferablyserotype C) or a cross-reactive antibody reactive with the APV, and amethod for serologically diagnosing an MPV infection of a mammalcomprising determining in a sample of the mammal the presence of across-reactive antibody that is also directed against an APV orcomponent thereof by reacting the sample with a proteinaceous moleculeor fragment thereof or an antigen derived from APV. Furthermore, theinvention provides the use of a diagnostic kit initially designed forAVP or AVP-antibody detection for diagnosing an MPV infection, inparticular for detecting the MPV infection in humans.

The invention also provides method for virologically diagnosing an APVinfection in a bird comprising determining in a sample of the bird thepresence of a viral isolate or component thereof by reacting the samplewith a cross-reactive nucleic acid derived from MPV or a cross-reactiveantibody reactive with the MPV, and a method for serologicallydiagnosing an APV infection of a bird comprising determining in a sampleof the bird the presence of a cross-reactive antibody that is alsodirected against an MPV or component thereof by reacting the sample witha proteinaceous molecule or fragment thereof or an antigen derived fromMPV.

Furthermore, the invention provides the use of a diagnostic kitinitially designed for MPV or MPV-antibody detection for diagnosing anAPV infection, in particular for detecting the APV infection in poultrysuch as a chicken, duck or turkey.

As the, with treatment, similar use can be made of the cross-reactivityfound, in particular when circumstances at hand make the use of the morehomologous approach less straightforward. Vaccinations that cannot wait,such as emergency vaccinations against MPV infections can for example beperformed with vaccine-preparations derived from APV (preferably type C)isolates when a more homologous MPV vaccine is not available, and, viceversa, vaccinations against APV infections can be contemplated withvaccine preparations derived from MPV. Also, reverse genetic techniquesmake it possible to generate chimeric APV-MPV virus constructs that areuseful as a vaccine, being sufficiently dissimilar to field isolates ofeach of the respective strains to be attenuated to a desirable level.Similar reverse genetic techniques will make it also possible togenerate chimeric paramyxovirus-Metapneumovirus constructs, such asRSV-MPV or PI3-MPV constructs for us in a vaccine preparation. Suchconstructs are particularly useful as a combination vaccine to combatrespiratory tract illnesses.

The invention thus provides a novel etiological agent, an isolatedessentially mammalian negative-sense single-stranded RNA virus (hereinalso called MPV) belonging to the subfamily Pneumovirinae of the familyParamyxoviridae but not identifiable as a classical Pneumovirus, andbelonging to the genus Metapneumovirus, and MPV-specific components orsynthetic analogues thereof Mammalian viruses resemblingMetapneumoviruses, i.e., Metapneumoviruses isolatable from mammals thatessentially function as natural host for the virus or cause disease inthe mammals, have until now not been found. Metapneumoviruses, ingeneral thought to be essentially restricted to poultry as natural hostor etiological agent of disease, are also known as avian Pneumoviruses.Recently, an APV isolate of duck was described (OR 2 801 607), furtherdemonstrating that APV infections are essentially restricted to birds asnatural hosts.

The invention provides an isolated mammalian Pneumovirus (herein alsocalled MPV) comprising a gene order and amino acid sequence distinctfrom that of the genus Pneumovirus and which is closely related andconsidering its phylogenetic relatedness likely belonging to the genusMetapneumovirus within the subfamily Pneumovirinae of the familyParamyxoviridae. Although until now, Metapneumoviruses have only beenisolated from birds, it is now shown that related, albeit materiallydistinct, viruses can be identified in other animal species such asmammals. Herein we show repeated isolation of MPV from humans, whereasno such reports exists for APV. Furthermore, unlike APV, MPV essentiallydoes not or only little replicates in chickens and turkeys where iteasily does in cynomolgus macaques. No reports have been found onreplication of APV in mammals. In addition, whereas specific anti-seraraised against MPV neutralize MPV, anti-sera raised against APV A, B orC do not neutralize MPV to the same extent, and this lack of fullcross-reactivity provides another proof for MPV being a differentMetapneumovirus. Furthermore, where APV and MPV share a similar geneorder, the G and SH proteins of MPV are largely different from the onesknown of APV in that they show no significant sequence homologies onboth the amino acid or nucleic acid level. Diagnostic assays todiscriminate between APV and MPV isolates or antibodies directed againstthese different viruses can advantageously be developed based on one orboth of these proteins (examples are IF, ELISA, neutralization assay,RT-PCR assay). However, also sequence and/or antigenic informationobtained from the more related N, P, M, F and L proteins of MPV andanalyses of sequence homologies with the respective proteins of APV, canalso be used to discriminate between APV and MPV. For example,phylogenetic analyses of sequence information obtained from MNV revealedthat MV and APV are two different viruses. In particular, thephylogenetic trees show that APV and MPV are two different lineages ofvirus. We have also shown that MPV is circulating in the humanpopulation for at least 50 years, therefore interspecies transmissionhas probably taken place at least 50 years ago and is not an everydayevent. Since MPV CPE was virtually indistinguishable from that caused byhRSV or hPIV-1 in tMK or other cell cultures, the MPV may have well goneunnoticed until now. tMK (tertiary monkey kidney cells, i.e., ME cellsin a third passage in cell culture) are preferably used due to theirlower costs in comparison to primary or secondary cultures. The CPE is,as well as with some of the classical Paramyxoviridae, characterized bysyncytium formation after which the cells showed rapid internaldisruption, followed by detachment of the cells from the monolayer. Thecells usually (but not always) displayed CPE after three passages ofvirus from original material, at day 10 to 14 post inoculation, somewhatlater than CPE caused by other viruses such as hRSV or hPIV-1.

Classically, as devastating agents of disease, paramyxoviruses accountfor many animal and human deaths worldwide each year. TheParamyxoviridae form a family within the order of Mononegavirales(negative-sense single-stranded RNA viruses), consisting of thesub-families Paramyxovirinae and Pneumovirinae. The latter sub-family isat present taxonomically divided in the genera Pneumovirus andMetapneumovirus. ¹ Human respiratory syncytial virus (hRSV), the typespecies of the Pneumovirus genus, is the single-most important cause oflower respiratory tract infections during infancy and early childhoodworldwide.² Other members of the Pneumovirus genus include the bovineand ovine respiratory syncytial viruses and pneumonia virus of mice(PVM).

Avian Pneumovirus (APV) also known as turkey rhinotracheitis virus(TRTV), the etiological agent of avian rhinotracheitis, an upperrespiratory tract infection of turkeys,³ is the sole member of therecently assigned Metapneumovirus genus, which, as said was until nownot associated with infections, or what is more, with disease ofmammals. Serological subgroups of APV can be differentiated on the basisof nucleotide or amino acid sequences of the G glycoprotein andneutralization tests using monoclonal antibodies that also recognize theG glycoprotein, Within subgroups A, B and D the G protein shows 98.5 to99.7% aa sequence identity within subgroups while between the subgroupsonly 31.2-38% aa identity is observed. See, for example, M. S. Collins,R. E. Gough, and D. J. Alexander, Antigenic differentiation of avianPneumovirus isolates using polyclonal antisera and mouse monoclonalantibodies, Avian Pathology, 1993, 22:469-479; J. K. A. Cook, B. V.Jones, M. M. Ellis, Antigenic differentiation of strains of turkeyrhinotracheitis virus using monoclonal antibodies, Avian Pathology,1993, 22:257-273; M. H. Bayon-Auboyer, et al., Nucleotide sequences ofthe F, L and G protein genes of two non-A/non-B avian Pneumoviruses(APV) reveal a novel APV subgroup, J. Gen. Virol. 2000, 81(Pt11):2723-33; B. S. Seal, Matrix protein gene nucleotide and predictedamino acid sequence demonstrate that the first US avian Pneumovirusisolate is distinct from European strains, Virus Res., 1998,58(1-2):45-52; M. H. Bayon-Auboyer, et al., Comparison of F-, G- andN-based RT-PCR protocols with conventional virological procedures forthe detection and typing of turkey rhinotracheitis virus, Arch. Virol.1999. 144(6):1091-109; K. Juhasz and A. J. Easton, Extensive sequencevariation in the attachment (G) protein gene of avian Pneumovirus:evidence for two distinct subgroups, J. Gen. Virol. 1994. 75 (Pt11):2873-80.

A further serotype of APV is provided in WO00/20600, which describes theColorado isolate of APV and compared it to known APV or TRT strains within vitro serum neutralization tests. First, the Colorado isolate wastested against monospecific polyclonal antisera to recognized TRTisolates. The Colorado isolate was not neutralized by monospecificantisera to any of the TRT strains. It was, however, neutralized by ahyperimmune antiserum raised against a subgroup A strain. This antiserumneutralized the homologous virus to a titer of 1:400 and the Coloradoisolate to a titer of 1:80. Using the above method, the Colorado isolatewas then tested against TRT monoclonal antibodies. In each case, thereciprocal neutralization titer was <10. Monospecific antiserum raisedto the Colorado isolate was also tested against TRT strains of bothsubgroups. None of the TRT strains tested were neutralized by theantiserum to the Colorado isolate.

The Colorado strain of APV does not protect SPF chicks against challengewith either a subgroup A or a subgroup B strain of TRT virus. Theseresults suggest that the Colorado isolate may be the first example of afurther serotype of avian Pneumovirus, as also suggested byBayon-Auboyer et al. (J. Gen. Vir. 81:2723-2733 (2000)).

In a preferred embodiment, the invention provides an isolated MPVtaxonomically corresponding to a (hereto unknown mammalian)Metapneumovirus comprising a gene order distinct from that of thePneumoviruses within the sub-family Pneumovirinae of the familyParamyxoviridae. The classification of the two genera is based primarilyon their gene constellation; Metapneumoviruses generally lacknon-structural proteins such NS1 or NS2 (see also Randhawa et al., J.Vir. 71:9849-9854 (1997), and the gene order is different from that ofPneumoviruses (RSV: ‘3-NS1-NS2-N-P-M-SH-G-F-M2-5’, APV:‘3-N-P-M-F-M2-SH-G-L-5’).^(4, 5, 6) MPV as provided by the invention ora virus isolate taxonomically corresponding therewith is upon EManalysis revealed by paramyxovirus-like particles. Consistent with theclassification, MPV or virus isolates phylogenetically corresponding ortaxonomically corresponding therewith are sensitive to treatment withchloroform; are cultured optimally on tMK cells or cells functionallyequivalent thereto and are essentially trypsine dependent in most cellcultures. Furthermore, the typical CPE and lack of hemagglutinatingactivity with most classically used red blood cells suggested that avirus as provided herein is, albeit only distantly, related to classicalPneumoviruses such as RSV. Although most paramyxoviruses havehemagglutinating activity, most of the Pneumoviruses do not.¹³ An MPVaccording to the invention also contains a second overlapping ORF (M2-2)in the nucleic acid fragment encoding the M2 protein, as in general mostother Pneumoviruses such as for example also demonstrated in Ahmadian etal., J. Gen. Vir. 80:2011-2016 (1999).

To find further viral isolates as provided by the invention it sufficesto test a sample, optionally obtained from a diseased animal or human,for the presence of a virus of the sub-family Pneumovirinae, and test athus obtained virus for the presence of genes encoding (functional) NS1or NS2 or essentially demonstrate a gene order that is different fromthat of Pneumoviruses such as RSV as already discussed above.Furthermore, a virus isolate phylogenetically corresponding and thustaxonomically corresponding with MPV may be found by cross-hybridizationexperiments using nucleic acid from a here provided MPV isolate, or inclassical cross-serology experiments using monoclonal antibodiesspecifically directed against and/or antigens and/or immunogensspecifically derived from an MPV isolate.

Newly isolated viruses are phylogenetically corresponding to and thustaxonomically corresponding to MPV when comprising a gene order and/oramino acid sequence sufficiently similar to our prototypic MPVisolate(s), or are structurally corresponding therewith, and show closerelatedness to the genus Metapneumovirus within the subfamilyPneumovirinae. The highest amino sequence homology, and defining thestructural correspondence on the individual protein level between MPVand any of the known other viruses of the same family to date (APVsubtype C) is for matrix 87%, for nucleoprotein 88%, for phosphoprotein68%, for fusion protein 81% and for parts of the polymerase protein56-64%, as can be deduced when comparing the sequences given in FIG. 6with sequences of other viruses, in particular of AVP-C. Individualproteins or whole virus isolates with, respectively, higher homology tothese mentioned maximum values are considered phylogeneticallycorresponding and thus taxonomically corresponding to MPV, and comprisea nucleic acid sequence structurally corresponding with a sequence asshown in FIG. 6. Herewith the invention provides a virusphylogenetically corresponding to the deposited virus. It should benoted that, similar to other viruses, a certain degree of variation isfound between different isolated essentially mammalian negative-sensesingle-stranded RNA virus isolates as provided herein. In phylogenetictrees, we have identified at least two genetic clusters of virusisolates based on comparative sequence analyses of parts of the L, M, Nand F genes. Based on nucleotide and amino-acid differences in the viralnucleic acid or amino acid sequences (the viral sequences), and inanalogy to other Pneumoviruses such as RSV, these MPV genotypesrepresent subtypes of MPV. Within each of the genetic clusters of MPVisolates, the percentage identity at the nucleotide level was found tobe 94-100 for L, 91-100 for M, 90-100 for N and 93-100 for F and at theamino acid level the percentage identity was found to be 91-100 for L,98-100 for M, 96-100 for N and 98-100 for F. A further comparison can befound in FIGS. 18 to 28. The minimum percentage identity at thenucleotide level for the entire group of isolated essentially mammaliannegative-sense single-stranded RNA virus as provided herein (MPVisolates) identified so far was 81 for L and M, 83 for N and 82 for F.At the amino acid level, this percentage was 91 for L and N, 94 for M,and 95 for F. The viral sequence of a MPV isolate or an isolated MPV Fgene as provided herein for example shows less than 81% nucleotidesequence identity or less than 82% (amino acid sequence identity withthe respective nucleotide or amino acid sequence of an APV-C fusion (F)gene as, for example, provided by Seal et al., Vir. Res. 66:139147(2000).

Also, the viral sequence of a MPV isolate or an isolated MPV L gene asprovided herein for example shows less than 61% nucleotide sequenceidentity or less than 63% amino acid sequence identity with therespective nucleotide or amino acid sequence of an APV-A polymerase geneas for example provided by Randhawa et al., J. Gen. Vir. 77:3047-3051(1996).

Sequence divergence of MPV strains around the world may be somewhathigher, in analogy with other viruses. Consequently, two potentialgenetic clusters are identified by analyses of partial nucleotidesequences in the N, M, F and L ORFs of 9 virus isolates. 90-100%nucleotide identity was observed within a cluster, and 81-88% identitywas observed between the clusters. Sequence information obtained on morevirus isolates confirmed the existence of two genotypes. Virus isolatened/00/01 as prototype of cluster A, and virus isolate ned/99/01 asprototype of cluster B have been used in cross-neutralization assays totest whether the genotypes are related to different serotypes orsubgroups. From these data we conclude that essentially mammalian virusisolates displaying percentage amino acid homology higher than 64 for L,87 for M, 88 for N, 68 for P, 81 for F 84 for M2-1 or 58 for M2-2 toisolate I-2614 may be classified as an isolated essentially mammaliannegative-sense single-stranded RNA virus as provided herein. Inparticular, those virus isolates in general that have a minimumpercentage identity at the nucleotide sequence level with a prototypeMPV isolate as provided herein of 81 for L and M, 83 for N and/or 82 forF are members of the group of MPV isolates as provided herein. At theamino acid level, these percentages are 91 for L and N, 94 for M, and/or95 for F. When the percentage amino acid sequence homology for a givenvirus isolate is higher than 90 for L and N, 93 for M, or 94 for F, thevirus isolate is similar to the group of MPV isolates displayed in FIG.5. When the percentage amino acid sequence homology for a given virusisolate is higher than 94 for L, 95 for N or 97 for M and F the virusisolate can be identified to belong to one of the genotype clustersrepresented in FIG. 5. It should be noted that these percentages ofhomology, by which genetic clusters are defined, are similar to thedegree of homology found among genetic clusters in the correspondinggenes of RSV.

In short, the invention provides an isolated essentially mammaliannegative-sense single-stranded RNA virus (MPV) belonging to thesub-family Pneumovirinae of the family Paramyxoviridae and identifiableas phylogenetically corresponding to the genus Metapneumovirus bydetermining a nucleic acid sequence of a suitable fragment of the genomeof the virus and testing it in phylogenetic tree analyses whereinmaximum likelihood trees are generated using 100 bootstraps and 3jumbles and finding it to be more closely phylogenetically correspondingto a virus isolate deposited as I-2614 with CNCM, Paris than it iscorresponding to a virus isolate of avian Pneumovirus (APV) also knownas turkey rhinotracheitis virus (TV), the etiological agent of avianrhinotracheitis.

Suitable nucleic acid genome fragments each useful for such phylogenetictree analyses are for example any of the RAP-PCR fragments 1 to 10 asdisclosed herein in the detailed description, leading to the variousphylogenetic tree analyses as disclosed herein in FIG. 4 or 5.Phylogenetic tree analyses of the nucleoprotein (N), phosphoprotein (P),matrix protein (M) and fusion protein (F) genes of MPV revealed thehighest degree of sequence homology with APV serotype C, the avianPneumovirus found primarily in birds in the United States.

In a preferred embodiment, the invention provides an isolatedessentially mammalian negative-sense single-stranded RNA virus (MPV)belonging to the sub-family Pneumovirinae of the family Paramyxoviridaeand identifiable as phylogenetically corresponding to the genusMetapneumovirus by determining a nucleic acid sequence of a suitablefragment of the genome of the virus and testing it in phylogenetic treeanalyses wherein maximum likelihood trees are generated using 100bootstraps and 3 jumbles and finding it to be more closelyphylogenetically corresponding to a virus isolate deposited as 1-2614with CNCM, Paris than it is corresponding to a virus isolate of avianPneumovirus (APV) also known as turkey rhinotracheitis virus (TRTV), theetiological agent of avian rhinotracheitis, wherein the suitablefragment comprises an open reading frame encoding a viral protein of thevirus.

A suitable open reading frame (ORF) comprises the ORF encoding the Nprotein. When an overall amino acid identity of at least 91%, preferablyof at least 95% of the analyzed N-protein with the N-protein of isolate1-2614 is found, the analyzed virus isolate comprises a preferred MPVisolate according to the invention. As shown, the first gene in thegenomic map of MPV codes for a 394 amino acid (aa) protein and showsextensive homology with the N protein of other Pneumoviruses. The lengthof the N ORF is identical to the length of the N ORF of APV-C (Table 5)and is smaller than those of other paramyxoviruses (Barr et al., 1991).Analysis of the amino acid sequence revealed the highest homology withAPV-C (88%), and only 7-11% with other paramyxoviruses (Table 6).

Barr et al. (1991) identified 3 regions of similarity between virusesbelonging to the order Mononegavirales: A, B and C (FIG. 8). Althoughsimilarities are highest within a virus family, these regions are highlyconserved between virus families. In all three regions MPV revealed 97%aa sequence identity with APV-C, 89% with APV-B, 92 with APV-A, and66-73% with RSV and PVM. The region between aa residues 160 and 340appears to be highly conserved among Metapneumoviruses and to a somewhatlesser extent the Pneumovirinae (Miyahara et al., 1992; Li et al., 1996;Barr et al., 1991). This is in agreement with MPV being aMetapneumovirus, this particular region showing 99% similarity with APVC.

Another suitable open reading frame (ORF) useful in phylogeneticanalyses comprises the ORF encoding the P protein. When an overall aminoacid-identity of at least 70%, preferably of at least 85% of theanalyzed P-protein with the P-protein of isolate I-2614 is found, theanalyzed virus isolate comprises a preferred MPV isolate according tothe invention. The second ORF in the genome map codes for a 294 aaprotein which shares 68% aa sequence homology with the P protein ofAPV-C, and only 22-26% with the P protein of RSV (Table 6). The P geneof MPV contains one substantial ORF and in that respect is similar to Pfrom many other paramyxoviruses (Reviewed in Lamb and Kolakofsky, 1996;Sedlmeier et al., 1998). In contrast to APV A and B and PVM and similarto RSV and APV-C the MPV P ORF lacks cysteine residues. Ling (1995)suggested that a region of high similarity between all Pneumoviruses (aa185-241) plays a role in either the RNA synthesis process or inmaintaining the structural integrity of the nucleocapsid complex. Thisregion of high similarity is also found in MPV (FIG. 9), specificallywhen conservative substitutions are taken in account, showing 100%similarity with APV-C, 93% with APV-A and B, and approximately 81% withRSV. The C-terminus of the MPV P protein is rich in glutamate residuesas has been described for APVs (Ling et al., 1995).

Another suitable open reading frame (ORF) useful in phylogeneticanalyses comprises the ORF encoding the M protein. When an overall aminoacid identity of at least 94%, preferably of at least 97% of theanalyzed M-protein with the M-protein of isolate I-2614 is found, theanalyzed virus isolate comprises a preferred MPV isolate according tothe invention. The third ORF of the MPV genome encodes a 254 aa protein,which resembles the M ORFs of other Pneumoviruses. The M ORF of MPV hasexactly the same size as the M ORFs of other Metapneumoviruses (Table 5)and shows high aa sequence homology with the matrix proteins of APV(76-87%) lower homology with those of RSV and PVM (37-38%) and 10% orless homology with those of other paramyxoviruses (Table 6). Easton(1997) compared the sequences of matrix proteins of all Pneumovirusesand found a conserved hexapeptide at residue 14 to 19 that is alsoconserved in MPV (FIG. 10). For RSV, PVM and APV small secondary ORFswithin or overlapping with the major ORF of M have been identified (52aa and 51 aa in bRSV, 75 aa in RSV, 46 aa in PVM and 51 aa in APV) (Yuet al., 1992; Easton et al., 1997; Samal et al., 1991; Satake et al.,1984). We noticed two small ORFs in the M ORF of MPV. One small ORF of54 aa residues was found within the major M ORF, starting at nucleotide2281 and one small ORF of 33 aa residues was found overlapping with themajor ORF of M starting at nucleotide 2893 (data not shown). Similar tothe secondary ORFs of RSV and APV there is no significant homologybetween these secondary ORFs and secondary ORFs of the otherPneumoviruses, and apparent start or stop signals are lacking. Inaddition, evidence for the synthesis of proteins corresponding to thesesecondary ORFs of APV and RSV has not been reported.

Another suitable open reading frame (ORF) useful in phylogeneticanalyses comprises the ORF encoding the F protein. When an overall aminoacid identity of at least 95%, preferably of at least 97% of theanalyzed F-protein with the F-protein of isolate I-2614 is found, theanalyzed virus isolate comprises a preferred MPV isolate according tothe invention. The F ORF of MPV is located adjacent to the M ORF, whichis characteristic for members of the Metapneumovirus genus. The F geneof MPV encodes a 539 aa protein, which is two aa residues longer than Fof APV-C (Table 5). Analysis of the aa sequence revealed 81% homologywith APV-C, 67% with APV-A and B, 33-39% with Pneumovirus F proteins andonly 10-18% with other paramyxoviruses (Table 6). One of the conservedfeatures among F proteins of paramyxoviruses, and also seen in MPV isthe distribution of cysteine residues (Morrison, 1988; Yu et al., 1991).The Metapneumoviruses share 12 cysteine residues in F1 (7 are conservedamong all paramyxoviruses), and two in F2 (I is conserved among allparamyxoviruses). Of the three potential N-linked glycosylation sitespresent in the F ORF of MPV, none are shared with RSV and two (position66 and 389) are shared with APV. The third, unique, potential N-linkedglycosylation site for MPV is located at position 206 (FIG. 11). Despitethe low sequence homology with other paramyxoviruses, the F protein ofMPV revealed typical fusion protein characteristics consistent withthose described for the F proteins of other Paramyxoviridae familymembers (Morrison, 1988). F proteins of Paramyxoviridae members aresynthesized as inactive precursors (F0) that are cleaved by host cellproteases which generate amino terminal F2 subunits and large carboxyterminal F1 subunits. The proposed cleavage site (Collins et al., 1996)is conserved among all members of the Paramyxoviridae family. Thecleavage site of MPV contains the residues RQSR. Both arginine (R)residues are shared with APV and RSV, but the glutamine (Q) and serine(S) residues are shared with other paramyxoviruses such as humanparainfluenza virus type 1, Sendai virus and morbilliviruses (data notshown). The hydrophobic region at the amino terminus of F1 is thought tofunction as the membrane fusion domain an shows high sequence similarityamong paramyxoviruses and morbilliviruses and to a lesser extent thePneumoviruses (Morrison, 1988). These 26 residues (position 137-163,FIG. 11) are conserved between MPV and APV.C, which is in agreement withthis region being highly conserved among the Metapneumoviruses (Nayloret al., 1998; Seal et al., 2000).

As is seen for the F2 subunits of APV and other paramyxoviruses, MPVrevealed a deletion of 22 aa residues compared with RSV (position107-128, FIG. 11). Furthermore, for RSV and APV, the signal peptide andanchor domain were found to be conserved within subtypes and displayedhigh variability between subtypes (Plows et al., 1995; Naylor et al.,1998). The signal peptide of MPV (aa 10-35, FIG. 11) at the aminoterminus of F2 exhibits some sequence similarity with APV-C (18 out of26 aa residues are similar) and less conservation with other APVs orRSV. Much more variability is seen in the membrane anchor domain at thecarboxy terminus of F1, although some homology is still seen with APV-C.

Another suitable open reading frame (ORF) useful in phylogeneticanalyses comprises the ORF encoding the M2 protein. When an overallamino acid identity of at least 85%, preferably of at least 90% of theanalyzed M2-protein with the M2-protein of isolate I-2614 is found, theanalyzed virus isolate comprises a preferred MPV isolate according tothe invention. M2 gene is unique to the Pneumovirinae and twooverlapping ORFs have been observed in all Pneumoviruses. The firstmajor ORF represents the M2-1 protein which enhances the processivity ofthe viral polymerase (Collins et al., 1995; Collins, 1996) and its readthrough of intergenic regions (Hardy et al., 1998; Fearns et al., 1999).The M2-1 gene for MPV, located adjacent to the F gene, encodes a 187 aaprotein (Table 5), and reveals the highest (84%) homology with M2-1 ofAPV-C (Table 6). Comparison of all Pneumovirus M2-1 proteins revealedthe highest conservation in the amino-terminal half of the protein(Collins et al., 1990; Zamora et al., 1992; Ahmadian et al., 1999),which is in agreement with the observation that MPV displays 100%similarity with APV-C in the first 80 aa residues of the protein (FIG.12A). The MPV M2-1 protein contains 3 cysteine residues located withinthe first 30 aa residues that are conserved among all Pneumoviruses.Such a concentration of cysteines is frequently found in zinc-bindingproteins (Ahmadian et al., 1991; Cuesta et al., 2000).

The secondary ORFs (M2-2) that overlap with the M2-1 ORFs ofPneumoviruses are conserved in location but not in sequence and arethought to be involved in the control of the switch between virus RNAreplication and transcription (Collins et al., 1985; Elango et al.,1985; Baybutt et al., 1987; Collins et al., 1990; Ling et al., 1992;Zamora et al., 1992; Alansari et al., 1994; Ahmadian et al., 1999;Bermingham et al., 1999). For MPV, the M2-2 ORF starts at nucleotide 512in the M2-1 ORF (FIG. 7), which is exactly the same start position asfor APV-C. The length of the M2-2 ORFs are the same for APV-C and MPV,71 aa residues (Table 5). Sequence comparison of the M2-2 ORF (FIG. 12B)revealed 56% aa sequence homology between MPV and APV-C and only 26-27%aa sequence homology between MPV and APV-A and B (Table 6).

Another suitable open reading frame (ORF) useful in phylogeneticanalyses comprises the ORF encoding the L protein. When an overall aminoacid identity of at least 91%, preferably of at least 95% of theanalyzed L-protein with the L-protein of isolate I-2614 is found, theanalyzed virus isolate comprises a preferred MPV isolate according tothe invention. In analogy to other negative strand viruses, the last ORFof the MPV genome is the RNA-dependent RNA polymerase component of thereplication and transcription complexes. The L gene of MPV encodes a2005 aa protein, which is 1 residue longer than the APV-A protein (Table5). The L protein of MPV shares 64% homology with APV-A, 42-44% withRSV, and approximately 13% with other paramyxoviruses (Table 6). Poch etal. (1989; 1990) identified six conserved domains within the L proteinsof non-segmented negative strand RNA viruses, from which domain IIIcontained the four core polymerase motifs that are thought to beessential for polymerase function. These motifs (A, B, C and D) are wellconserved in the MPV L protein: in motifs A, B and C: MPV shares 100%similarity with all Pneumoviruses and in motif D MPV shares 100%similarity with APV and 92% with RSVs. For the entire domain III (aa625-847 in the L ORF), MPV shares 83% identity with APV, 67-68% with RSVand 26-30% with other paramyxoviruses (FIG. 15). In addition to thepolymerase motifs the Pneumovirus L proteins contain a sequence whichconforms to a consensus ATP binding motif K(X)₂₁GEGAGN(X)₂₀K (SEQ IDNO:105) (Stec, 1991). The MPV L ORF contains a similar motif as APV, inwhich the spacing of the intermediate residues is off by one:K(x)₂₂GEGAGN(X)₁₉ K (SEQ ID NO:106).

A much preferred suitable open reading frame (ORF) useful inphylogenetic analyses comprises the ORF encoding the SH protein. When anoverall amino acid identity of at least 30%, preferably of at least 50%,more preferably of at least 75% of the analyzed SH-protein with theSH-protein of isolate I-2614 is found, the analyzed virus isolatecomprises a preferred MPV isolate according to the invention. The genelocated adjacent to M2 of MPV encodes a 183 aa protein (FIG. 7).Analysis of the nucleotide sequence and its deduced amino acid sequencerevealed no discernible homology with other RNA virus genes or geneproducts. The SH ORF of MPV is the longest SH ORF known to date (Table5). The composition of the aa residues of the SH ORF is relativelysimilar to that of APV, RSV and PVM, with a high percentage of threonineand serine (22%, 18%, 19%, 20.0%, 21% and 28% serine/threonine contentfor MPV, APV, RSV A, RSV B, bRSV and PVM respectively). The SH ORF ofMPV contains ten cysteine residues, whereas APV SH contains 16 cysteineresidues. All Pneumoviruses have similar numbers of potentialN-glycosylation sites (MPV 2, APV 1, RSV 2, bRSV 3, PVM 4).

The hydrophobicity profiles for the MPV SH protein and SH of APV and RSVrevealed similar structural characteristics (FIG. 13B). The SH ORFs ofAPV and MPV have a hydrophilic N-terminus (aa 1-30), a centralhydrophobic domain (aa 30-53) which can serve as a potential membranespanning domain, a second hydrophobic domain around residue 160 and ahydrophilic C-terminus. In contrast, RSV SH appears to lack theC-terminal half of the APV and MPV ORFs. In all Pneumovirus SH proteinsthe hydrophobic domain is flanked by basic amino acids, which are alsofound in the SH ORF for MPV (aa 29 and 64).

Another much preferred suitable open reading frame (ORF) useful-inphylogenetic analyses comprises the ORF encoding the G protein. When anoverall amino acid identity of at least 30%, preferably of at least 50%,more preferably of at least 75% of the analyzed G-protein with theG-protein of isolate I-2614 is found, the analyzed virus isolatecomprises a preferred MPV isolate according to the invention. The G ORFof MPV is located adjacent to the SH gene and encodes a 236 amino acidprotein. A secondary small ORF is found immediately following this ORF,potentially coding for 68 aa residues (pos. 6973-7179,), but lacking astart codon. A third major ORF, in a different reading frame, of 194 aaresidues (fragment 4, FIG. 7) is overlapping with both of these ORFs,but also lacks a start codon (nucleotide 6416-7000). This major ORF isfollowed by a fourth ORF in the same reading frame (nt 7001-7198),possibly coding for 65 aa residues but again lacking a start codon.Finally, a potential ORF of 97 aa residues (but lacking a start codon)is found in the third reading frame (nt 6444-6737, FIG. 1). Unlike thefirst ORF, the other ORFs do not have apparent gene start or gene endsequences (see below). Although the 236 aa residue G ORF probablyrepresents at least a part of the MPV attachment protein it cannot beexcluded that the additional coding sequences are expressed as separateproteins or as part of the attachment protein through some RNA editingevent. It should be noted that for APV and RSV no secondary ORFs afterthe primary G ORF have been identified but that both APV and RSV havesecondary ORFs within the major ORF of G. However, evidence forexpression of these ORFEs is lacking and there is no homology betweenthe predicted aa sequences for different viruses (Ling et al., 1992).The secondary ORFs in MPV G do not reveal characteristics of other Gproteins and whether the additional ORFs are expressed requires furtherinvestigation. BLAST analyses with all four ORFs revealed no discerniblehomology at the nucleotide or aa sequence level with other known virusgenes or gene products. This is in agreement with the low sequencehomologies found for other G proteins such as hRSV A and B (53%)(Johnson et al., 1987) and APV A and B (38%) (Juhasz et al., 1994).Whereas most of the MPV ORFs resemble those of APV both in length andsequence, the G ORF of MPV is considerably smaller than the G ORF of APV(Table 5). The aa sequence revealed a serine and threonine content of34%, which is even higher than the 32% for RSV and 24% for APV. The GORF also contains 8.5% proline residues, which is higher than the 8% forRSV and 7% for APV. The unusual abundance of proline residues in the Gproteins of APV, RSV and MPV has also been observed in glycoproteins ofmucinous origin where it is a major determinant of the proteins threedimensional structure (Collins et al., 1983; Wertz et al., 1985;Jentoft, 1990). The number of potential N-linked glycosylation sites inG of MPV is similar to other Pneumoviruses: MPV has 5, whereas hRSV has7, bRSV has 5, and APV has 3 to 5.

The predicted hydrophobicity profile of MPV G revealed characteristicssimilar to the other Pneumoviruses. The amino-terminus contains ahydrophilic region followed by a short hydrophobic area (aa 33-53) and amainly hydrophilic carboxy terminus (FIG. 14B). This overallorganization is consistent with that of an anchored type IItransmembrane protein and corresponds well with these regions in the Gprotein of APV and RSV. The G ORF of MPV contains only 1 cysteineresidue in contrast to RSV and APV (5 and 20, respectively).

According to classical serological analyses as for example known from R.I. B. Francki, C. M. Fauquet, D. L. Knudson, and F. Brown,Classification and nomenclature of viruses, Fifth report of theinternational Committee on Taxonomy of Viruses, Arch Virol. 1991,Supplement 2:140-144, an MPV isolate is also identifiable as belongingto a serotype as provided herein, being defined on the basis of itsimmunological distinctiveness, as determined by quantitativeneutralization with animal antisera (obtained from for example ferretsor guinea pigs as provided in the detailed description). Such a serotypehas either no cross-reaction with others or shows a homologous-toheterologous titer ratio >16 in both directions. If neutralization showsa certain degree of cross-reaction between two viruses in either or bothdirections (homologous-to-heterologous tier ration of eight or 16),distinctiveness of serotype is assumed if substantialbiophysical/biochemical differences of DNAs exist. If neutralizationshows a distinct degree of cross-reaction between two viruses in eitheror both directions (homologous-to-heterologous tier ration of smallerthan eight), identity of serotype of the isolates under study isassumed. As said, useful prototype isolates, such as isolate I-2614,herein also known as MPV isolate 00-1, are provided herein.

A further classification of a virus as an isolated essentially mammaliannegative-sense single-stranded RNA virus as provided herein can be madeon the basis of homology to the G and/or SH proteins. Where in generalthe overall amino acid sequence identity between APV (isolated frombirds) and MPV (isolated from humans) N, P, M, F, M2 and L ORFs was 64to 88 percent, and nucleotide sequence homology was also found betweenthe non-coding regions of the APV and MPV genomes, essentially nodiscernible amino acid sequence homology was found between two of theORFs of the human isolate (MPV) and any of the ORFs of otherparamyxoviruses. The amino acid content, hydrophobicity profiles andlocation of these ORFs in the viral genome show that they represent Gand SH protein analogues. The sequence homology between APV and MPV,their similar genomic organization (3′-N-P-M-F-M2-SH-G-L5′) as well asphylogenetic analyses provide further evidence for the proposedclassification of MPV as the first mammalian Metapneumovirus. New MPVisolates are for thus example identified as such by virus isolation andcharacterization on tMK or other cells, by RT-PCR and/or sequenceanalysis followed by phylogenetic tree analyses, and by serologictechniques such as virus neutralization assays, indirectimmunofluorescence assays, direct immunofluorescence assays, FACsanalyses or other immunological techniques.

Preferably these techniques are directed at the SH and/or G proteinanalogues.

For example the invention provides herein a method to identify furtherisolates of MPV as provided herein, the method comprising inoculating anessentially MPV-uninfected or specific-pathogen-free guinea pig orferret (in the detailed description the animal is inoculatedintranasally but other ways of inoculation such as intramuscular orintradermal inoculation, and using another experimental animal, is alsofeasible) with the prototype isolate I-2614 or related isolates. Seraare collected from the animal at day zero, two weeks and three weekspost inoculation. The animal specifically seroconverted as measured invirus neutralization (VN) assay and indirect IFA against the respectiveisolate I-2614 and the sera from the seroconverted animal are used inthe immunological detection of the further isolates.

As an example, the invention provides the characterization of a newmember in the family of Paramyxoviridae, a human Metapneumovirus orMetapneumovirus-like virus (since its final taxonomy awaits discussionby a viral taxonomy committee the MPV is herein for example described astaxonomically corresponding to APV) (MPV) which may cause severe RTI inhumans. The clinical signs of the disease caused by MPV are essentiallysimilar to those caused by hRSV, such as cough, myalgia, vomiting,fever, bronchiolitis or pneumonia, possible conjunctivitis, orcombinations thereof. As is seen with hRSV-infected children, especiallyvery young children may require hospitalization. As an example an MPVwhich was deposited Jan. 19, 2001 as I-2614 with CNCM, InstitutePasteur, Paris or a virus isolate phylogenetically correspondingtherewith is herewith provided. Therewith, the invention provides avirus comprising a nucleic acid or functional fragment phylogeneticallycorresponding to a nucleic acid sequence shown in FIGS. 6 a, 6 b, 6 c,or structurally corresponding therewith. In particular the inventionprovides a virus characterized in that after testing it in phylogenetictree analyses wherein maximum likelihood trees are generated using 100bootstraps and 3 jumbles it is found to be more closely phylogeneticallycorresponding to a virus isolate deposited as I-2614 with CNCM, Paristhan it is related to a virus isolate of avian Pneumovirus (APV) alsoknown as turkey rhinotracheitis virus (TRTV), the etiological agent ofavian rhinotracheitis. It is particularly useful to use an AVP-C virusisolate as outgroup in the phylogenetic tree analyses, it being theclosest relative, albeit being an essentially non-mammalian virus.

We propose the new human virus to be named human Metapneumovirus orMetapneumovirus-like virus (MPV) based on several observations. EManalysis revealed paramyxovirus-like particles. Consistent with theclassification, MPV appeared to be sensitive to treatment withchloroform. WPV is cultured optimal on tMK cells and is trypsinedependent. The clinical symptoms caused by MPV as well as the typicalCPE and lack of hemagglutinating activity suggested that this virus isclosely related to hRSV. Although most paramyxoviruses havehemagglutinating activity, most of the Pneumoviruses do not.¹³

As an example, the invention provides a not previously identifiedparamyxovirus from nasopharyngeal aspirate samples taken from 28children suffering from severe RTI. The clinical symptoms of thesechildren were largely similar to those caused by hRSV. Twenty-seven ofthe patients were children below the age of five years and half of thesewere between 1 and 12 months old. The other patient was 18 years old.All individuals suffered from upper RTI, with symptoms ranging fromcough, myalgia, vomiting and fever to bronchiolitis and severepneumonia. The majority of these patients were hospitalized for one totwo weeks.

The virus isolates from these patients had the paramyxovirus morphologyin negative contrast electron microscopy but did not react with specificantisera against known human and animal paramyxoviruses. They were allclosely related to one another as determined by indirectimmunofluorescence assays (IFA) with sera raised against two of theisolates. Sequence analyses of nine of these isolates revealed that thevirus is somewhat related to APV. Based on virological data, sequencehomology as well as the genomic organization we propose that the virusis a member of Metapneumovirus genus. Serological surveys showed thatthis virus is a relatively common pathogen since the seroprevalence inthe Netherlands approaches 100% of humans by the age of five years.Moreover, the seroprevalence was found to be equally high in seracollected from humans in 1958, indicating this virus has beencirculating in the human population for more than 40 years. Theidentification of this proposed new member of the Metapneumovirus genusnow also provides for the development of means and methods fordiagnostic assays or test kits and vaccines or serum or antibodycompositions for viral respiratory tract infections, and for methods totest or screen for antiviral agents useful in the treatment of MPVinfections.

To this extent, the invention provides among others an isolated orrecombinant nucleic acid or virus-specific functional fragment thereofobtainable from a virus according to the invention. In particular, theinvention provides primers and/or probes suitable for identifying an MPVnucleic acid.

Furthermore, the invention provides a vector comprising a nucleic acidaccording to the invention. To begin with, vectors such as plasmidvectors containing (parts of) the genome of MPV, virus vectorscontaining (parts of) the genome of MPV. (For example, but not limitedto other paramyxoviruses, vaccinia virus, retroviruses, baculovirus), orMPV containing (parts of) the genome of other viruses or other pathogensare provided. Furthermore, a number of reverse genetics techniques havebeen described for the generation of recombinant negative strandviruses, based on two critical parameters. First, the production of suchvirus relies on the replication of a partial or full-length copy of thenegative sense viral RNA (vRNA) genome or a complementary copy thereof(cRNA). This vRNA or cRNA can be isolated from infectious virus,produced upon in vitro transcription, or produced in cells upontransfection of nucleic acids. Second, the production of recombinantnegative strand virus relies on a functional polymerase complex.Typically, the polymerase complex of Pneumoviruses consists of N, P, Land possibly M2 proteins, but is not necessarily limited thereto.Polymerase complexes or components thereof can be isolated from virusparticles, isolated from cells expressing one or more of the components,or produced upon transfection of specific expression vectors.

Infectious copies of MPV can be obtained when the above mentioned vRNA,cRNA, or vectors expressing these RNAs are replicated by the abovementioned polymerase complex.^(16, 17, 18, 19, 20, 21, 22) For thegeneration of minireplicons or, a reverse genetics system for generatinga full-length copy comprising most or all of the genome of MPV itsuffices to use 3′ end and/or 5′ end nucleic acid sequences obtainablefrom for example APV (Randhawa et al., 1997) or MPV itself.

Also, the invention provides a host cell comprising a nucleic acid or avector according to the invention. Plasmid or viral vectors containingthe polymerase components of MPV (presumably N, P, L and M2, but notnecessarily limited thereto) are generated in prokaryotic cells for theexpression of the components in relevant cell types (bacteria, insectcells, eukaryotic cells). Plasmid or viral vectors containingfull-length or partial copies of the MPV genome will be generated inprolaryotic cells for the expression of viral nucleic acids in vitro orin vivo. The latter vectors may contain other viral sequences for thegeneration of chimeric viruses or chimeric virus proteins, may lackparts of the viral genome for the generation of replication defectivevirus, and may contain mutations, deletions or insertions for thegeneration of attenuated viruses.

Infectious copies of MPV (being wild type, attenuated,replication-defective or chimeric) can be produced upon co-expression ofthe polymerase components according to the state-of-the-art technologiesdescribed above.

In addition, eukaryotic cells, transiently or stably expressing one ormore full-length or partial MPV proteins can be used. Such cells can bemade by transfection (proteins or nucleic acid vectors), infection(viral vectors) or transduction (viral vectors) and may be useful forcomplementation of mentioned wild type, attenuated,replication-defective or chimeric viruses.

A chimeric virus may be of particular use for the generation ofrecombinant vaccines protecting against two or moreviruses.^(23, 24, 26) For example, it can be envisaged that a MPV virusvector expressing one or more proteins of RSV or a RSV vector expressingone or more proteins of MPV will protect individuals vaccinated withsuch vector against both virus infections. A similar approach can beenvisaged for PI3 or other paramyxoviruses. Attenuated andreplication-defective viruses may be of use for vaccination purposeswith live vaccines as has been suggested for other viruses.^(25, 26)

In a preferred embodiment, the invention provides a proteinaceousmolecule or Metapneumovirus-specific viral protein or functionalfragment thereof encoded by a nucleic acid according to the invention.Useful proteinaceous molecules are for example derived-from any of thegenes or genomic fragments derivable from a virus according to theinvention. Such molecules, or antigenic fragments thereof, as providedherein, are for example useful in diagnostic methods or kits and inpharmaceutical compositions such as sub-unit vaccines. Particularlyuseful are the F, SH and/or G protein or antigenic fragments thereof forinclusion as antigen or subunit immunogen, but inactivated whole viruscan also be used. Particularly useful are also those proteinaceoussubstances that are encoded by recombinant nucleic acid fragments thatare identified for phylogenetic analyses, of course preferred are thosethat are within the preferred bounds and metes of ORFs useful inphylogenetic analyses, in particular for eliciting MPV specificantibodies, whether in vivo (e.g., for protective purposes or forproviding diagnostic antibodies) or in vitro (e.g., by phage displaytechnology or another technique useful for generating syntheticantibodies).

Also provided herein are antibodies, be it natural polyclonal ormonoclonal or synthetic (e.g., (phage) library-derived bindingmolecules) antibodies that specifically react with an antigen comprisinga proteinaceous molecule or MPV-specific functional fragment thereofaccording to the invention. Such antibodies are useful in a method foridentifying a viral isolate as an MPV comprising reacting the viralisolate or a component thereof with an antibody as provided herein. Thiscan for example be achieved by using purified or non-purified MPV orparts thereof (proteins, peptides) using EIASA, RIA, FACS or similarformats of antigen detection assays (Current Protocols in Immunology).Alternatively, infected cells or cell cultures may be used to identifyviral antigens using classical immunofluorescence or immunohistochemicaltechniques.

Other methods for identifying a viral isolate as a MPV comprise reactingthe viral isolate or a component thereof with a virus specific nucleicacid according to the invention, in particular where the mammalian viruscomprises a human virus.

In this way the invention provides a viral isolate identifiable with amethod according to the invention as a mammalian virus taxonomicallycorresponding to a negative-sense single-stranded RNA virus identifiableas likely belonging to the genus Metapneumovirus within the sub-familyPneumovirinae of the family Paramyxoviridae.

The method is useful in a method for virologically diagnosing an MPVinfection of a mammal, the method for example comprising determining ina sample of the mammal the presence of a viral isolate or componentthereof by reacting the sample with a nucleic acid or an antibodyaccording to the invention. Examples are further given in the detaileddescription, such as the use of PCR (or other amplification orhybridization techniques well known in the art) or the use ofimmunofluorescence detection (or other immunological techniques known inthe art).

The invention also provides a method for serologically diagnosing a MPVinfection of a mammal comprising determining in a sample of the mammalthe presence of an antibody specifically directed against a MPV orcomponent thereof by reacting the sample with a proteinaceous moleculeor fragment thereof or an antigen according to the invention.

Methods and means provided herein are particularly useful in adiagnostic kit for diagnosing a MPV infection, be it by virological orserological diagnosis. Such kits or assays may for example comprise avirus, a nucleic acid, a proteinaceous molecule or fragment thereof, anantigen and/or an antibody according to the invention. Use of a virus, anucleic acid, a proteinaceous molecule or fragment thereof an antigenand/or an antibody according to the invention is also provided for theproduction of a pharmaceutical composition, for example, for thetreatment or prevention of MPV infections and/or for the treatment orprevention of respiratory tract illnesses, in particular in humans.Attenuation of the virus can be achieved by established methodsdeveloped for this purpose, including but not limited to the use ofrelated viruses of other species, serial passages through laboratoryanimals or/and tissue/cell cultures, site directed mutagenesis ofmolecular clones and exchange of genes or gene fragments between relatedviruses.

A pharmaceutical composition comprising a virus, a nucleic acid, aproteinaceous molecule or fragment thereof, an antigen and/or anantibody according to the invention can for example be used in a methodfor the treatment or prevention of a MPV infection and/or a respiratoryillness comprising providing an individual with a pharmaceuticalcomposition according to the invention. This is most useful when theindividual comprises a human, especially when the human is below 5 yearsof age, since such infants and young children are most likely to beinfected by a human MPV as provided herein. Generally, in the acutephase patients will suffer from upper respiratory symptoms predisposingfor other respiratory and other diseases. Also lower respiratoryillnesses may occur, predisposing for more and other serious conditions.

The invention also provides method to obtain an antiviral agent usefulin the treatment of respiratory tract illness comprising establishing acell culture or experimental animal comprising a virus according to theinvention, treating the culture or animal with an candidate antiviralagent, and determining the effect of the agent on the virus or itsinfection of the culture or animal. An example of such an antiviralagent comprises a MPV-neutralizing antibody, or functional componentthereof, as provided herein, but antiviral agents of other nature areobtained as well. The invention also provides use of an antiviral agentaccording to the invention for the preparation of a pharmaceuticalcomposition, in particular for the preparation of a pharmaceuticalcomposition for the treatment of respiratory tract illness, especiallywhen caused by an MPV infection, and provides a pharmaceuticalcomposition comprising an antiviral agent according to the invention,useful in a method for the treatment or prevention of an MPV infectionor respiratory illness, the method comprising providing an individualwith such a pharmaceutical composition.

The invention is further explained in the detailed description withoutlimiting it thereto. Deposit of Biological Material

Mammalian Metapneumovirus isolate NL/1/00 “MPV-isolate 00-1” has beendeposited with the international depository authority CollectionNationale de Cultures de Microorganismes (CNCM) as deposit accessionnumber I-2614. The address of the CNCM is Institut Pasteur, 26, Rue duDocteur Roux, F-75724 Paris Cedex 15, France. The deposits were receivedon Jan. 19, 2001.

BRIEF SUMMARY OF THE DRAWINGS

FIG. 1A comprises table 1: Percentage homology found between the aminoacid sequence of isolate 00-1 and other members of the Pneumovirinae.Percentages (×100) are given for the amino acid sequences of N, P, M, Fand two RAP-PCR fragments in L (8 and 9/10). Accession numbers used forthe analyses are described in the materials and methods section.

FIG. 1B comprises table 2: Seroprevalence of MPV in humans categorizedby age group using immunofluorescence and virus neutralization assays.

FIG. 2: Schematic representation of the genome of APV with the locationand size of the fragments obtained with RAP-PCR and RT-PCR on virusisolate 00-1. Fragments 1 to 10 were obtained using RAP-PCR. Fragment Awas obtained with a primer in RAP-PCR fragment 1 and 2 and a primerdesigned based on alignment of leader and trailer sequences of APV andRSVS. Fragment B was obtained using primers designed in RAP-PCRfragments 1 and 2 and RAP-PCR fragment 3. Fragment C was obtained withprimers designed in RAP-PCR fragment 3 and RAP-PCR fragments 4, 5, 6 and7.

For all phylogenetic trees, (FIGS. 3-5) DNA sequences were aligned usingthe ClustalW software package and maximum likelihood trees weregenerated using the DNA-ML software package of the Phylip 3.5 programusing 100 bootstraps and 3 jumbles.¹⁵ Previously published sequencesthat were used for the generation of phylogenetic trees are availablefrom Genbank under accessions numbers: For all ORFs: hRSV: NC001781;bRSV: NC001989; For the F ORF: PVM, D11128; APV-A, D00850; APV-B,Y14292; APV-C, AF187152; For the N ORF: PVM, D10331; APV-A, U39295;APV-B, U39296; APV-C, AF176590; For the M ORF: PMV, U66893; APV-A,X58639; APV-B, U37586; APV-C, AF262571; For the P ORF: PVM, 09649;APV-A, U22110, APV-C, AF176591. Phylogenetic analyses for the ninedifferent virus isolates of MPV were performed with APV strain C asoutgroup. Abbreviations used in figures: hRSV: human RSV; bRSV: bovineRSV, PVM: pneumonia virus of mic⁻; APV-A, B, and C: avian Pneumovirustype A, B and C.

FIG. 3 Comparison of the N (SEQ ID NOS:1-7), P (SEQ ID NOS:8-13), M (SEQID NOS:14-20) and F (SEQ ID NOS:21-27) ORFs of members of the subfamilyPneumovirinae and virus isolate 00-1. The alignment shows the amino acidsequence of the complete N (SEQ ID NO:1), P (SEQ ID NO:8), M (SEQ IDNO:14) and F (SEQ ID NO:21) proteins and partial L proteins (SEQ IDNO:28 and SEQ ID NO:32) of virus isolate 00-1. Amino acids that differbetween isolate 00-1 and the other viruses are shown, identical aminoacids are represented by periods, gaps are represented as dashes.Numbers correspond to amino acid positions in the proteins. Accessionnumbers used for the analyses are described in the materials and methodssection. APV-A, B or C: Avian Pneumovirus type A (SEQ ID NO:2, SEQ IDNO:9, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:29, SEQ ID NO:33), B (SEQ IDNO:3, SEQ ID NO:15, SEQ ID NO:23) or C (SEQ ID NO:4, SEQ ID NO:10, SEQID NO:17, SEQ ID NO:24), b- or hRSV: bovine (SEQ ID NO:5, SEQ ID NO:11,SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:34) or human (SEQ IDNO:6, SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:31, SEQ IDNO:35) respiratory syncytial virus, PVM: pneumonia virus of mice (SEQ IDNO:7, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:27). L8: fragment 8 obtainedwith RAP-PCR located in L, L9/10: consensus of fragment 9 and 10obtained with RAP-PCR, located in L. For the P alignment, no APV-Bsequence was available from the Genebank. For the L alignment only bRSV,hRSV and APV-A sequences were available.

FIG. 4: Phylogenetic analyses of the N, P, M, and F ORFs of members ofthe genus Pneumovirinae and virus isolate 00-1. Phylogenetic analysiswas performed on viral sequences from the following genes: F (panel A),N (panel B), M (panel C), and P (panel D). The phylogenetic trees arebased on maximum likelihood analyses using 100 bootstraps and 3 jumbles.The scale representing the number of nucleotide changes is shown foreach tree.

FIG. 5: Phylogenetic relationship for parts of the F (panel A), N (panelB), M (panel C) and L (panel D) ORFs of nine of the primary MPV isolateswith APV-C, its closest relative genetically. The phylogenetic trees arebased on maximum likelihood analyses. The scale representing the numberof nucleotide changes is shown for each tree. Accession numbers forAPV-C: panel A-D00850; panel B: U39295; panel C: X58639; and panel D:U65312.

FIG. 6A: Nucleotide (SEQ ID NO:36) and amino acid (SEQ ID NO:37, SEQ IDNO:8, SEQ ID NO:14, SEQ ID NO:21) sequence information from the 3′ endof the genome of MPV isolate 00-1.ORFs are given. N: ORF fornucleoprotein; P: ORF for phosphoprotein; M: ORF for matrix protein; F:ORF for fusion protein; GE: gene end; GS: gene start.

FIGS. 6B and 6C: Nucleotide and amino acid sequence information fromobtained fragments in the polymerase gene (L) of MPV isolates 00-1.Positioning of the fragments in L is based on protein homologies withAPV-C (accession number U65312). The translated fragment 8 (FIG. 6B)(SEQ ID NO:38 and SEQ ID NO:39) is located at amino acid number 8 to243, and the consensus of fragments 9 and 10 (FIG. 6C) (SEQ ID NO:40 andSEQ ID NO:41) is located at amino acid number 1358 to 1464 of the APV-CL ORF.

FIG. 7: Genomic map of NPV isolate 00-1. The nucleotide positions of thestart and stop codons are indicated under each ORF. The double lineswhich cross the L ORF indicate the shortened representation of the Lgene. The three reading frames below the map indicate the primary G ORF(nt 6262-6972) and overlapping potential secondary ORFS.

FIG. 8: Alignment of the predicted amino acid sequence of thenucleoprotein of MPV (SEQ ID NO:1) with those of other Pneumoviruses(SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:42, SEQ ID NO:6, SEQID NO:5, SEQ ID NO:7). The conserved regions identified by Barr (1991)are represented by boxes and labeled A, B, and C. The conserved regionamong Pneumoviruses (Li, 1996) is shown gray shaded. Gaps arerepresented by dashes, periods indicate the positions of identical aminoacid residues compared to MPV.

FIG. 9: Amino acid sequence comparison of the phosphoprotein of MPV (SEQID NO:8) with those of other Pneumoviruses (SEQ ID NO:10, SEQ ID NO:43,SEQ ID NO:9, SEQ ID NO:44, SEQ ID NO:12, SEQ ID NO:11, SEQ ID NO:13).The region of high similarity (Ling, 1995) is boxed, and the glutamaterich region is grey shaded. Gaps are represented by dashes and periodsindicate the position of identical amino acid residues compared to MPV.

FIG. 10: Comparison of the deduced amino acid sequence of the matrixprotein of MPV (SEQ ID NO:14) with those of other Pneumoviruses (SEQ IDNO:17, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:45, SEQ ID NO:19, SEQ IDNO:18, SEQ ID NO:20). The conserved hexapeptide sequence (Easton, 1997)is grey shaded. Gaps are represented by dashes and periods indicate theposition of identical amino acid residues relative to MPV.

FIG. 11: Alignment of the predicted amino acid sequence of the fusionprotein of MPV (SEQ ID NO:21) with those of other Pneumoviruses (SEQ IDNO:24, SEQ ID NO:23, SEQ ID NO:22, SEQ ID NO:46, SEQ ID NO:26, SEQ IDNO:25, SEQ ID NO:27). The conserved cysteine residues are boxed,N-linked glycosylation sites are underlined, the cleavage site of F0 isdouble underlined, the fusion peptide, signal peptide and membraneanchor domain are shown grey shaded. Gaps are represented by dashes andperiods indicate the position of identical amino acids relative to MPV.

FIG. 12: Comparison of amino acid sequence of the M2 ORFs of MPV withthose of other Pneumoviruses. The alignment of M2-1 ORFs is shown inpanel A (SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54), with the conservedamino terminus (Collins, 1990; Zamora, 1999) shown grey shaded. Thethree conserved cysteine residues are printed bold face and indicated by#. The alignment of M2-2 ORFs is shown in panel B (SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62). Gaps are represented by dashes and periodsindicate the position of identical amino acids relative to MPV.

FIG. 13: Amino acid sequence analyses of the SH ORF of MPV. (A) Aminoacid sequence of the SH ORF of MPV (SEQ ID NO:63), with the serine andthreonine residues grey shaded, cysteine residues in bold face and thehydrophobic region double underlined. Potential N-linked glycosylationsites are single underlined. Numbers indicate the positions of the basicamino acids flanking the hydrophobic domain. (B) Alignment of thehydrophobicity plots of the SH proteins of MPV, APV-A and hRSV-B. Theprocedure of Kyte and Doolittle (1982) was used with a window of 17amino acids. Arrows indicate a strong hydrophobic domain. Positionswithin the ORF are given on the X-axis.

FIG. 14: Amino acid sequence analyses of the G ORF of MPV. (A) Aminoacid sequence of the G ORF of MPV (SEQ ID NO:64), with serine, threonineand proline residues grey shaded, the cysteine residue is in bold faceand the hydrophobic region double underlined. The potential N-linkedglycosylation sites are single underlined. (B) Alignment of thehydrophobicity plots of the G proteins of MPV, APV-A and hRSV-B. Theprocedure of Kyte and Doolittle (1982) was used with a window of 17amino acids. Arrows indicate the hydrophobic region, and positionswithin the ORF are given at the X-axis.

FIG. 15: Comparison of the amino acid sequences of a conserved domain ofthe polymerase gene of MPV (SEQ ID NO:65) and other paramyxoviruses (SEQID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75). DomainIII is shown with the four conserved polymerase motifs (A, B, C, D) indomain III (Poch 1998, 1999) boxed. Gaps are represented by dashes andperiods indicate the position of identical amino acid residues relativeto MPV. hPIV3: human parainfluenza virus type 3; SV: sendai virus;hPIV-2: human parainfluenza virus type 2; NDV: New castle disease virus;MV: measles virus; nipah: Nipah virus.

FIG. 16: Phylogenetic analyses of the M2-1 and L ORFs of MPV andselected paramyxoviruses. The M2-1 ORF was aligned with the M2-1 ORFs ofother members of the genus Pneumovirinae (A) and the L ORF was alignedwith L ORFs members of the genus Pneumovirinae and selected otherparamyxoviruses as described in the legends of FIG. 15(B). Phylogenetictrees were generated by maximum likelihood analyses using 100 bootstrapsand 3 jumbles. The scale representing the number of nucleotide changesis shown for each tree. Numbers in the trees represent bootstrap valuesbased on the consensus trees.

FIG. 17: Noncoding sequences of hMPV isolate 00-1. (A) The noncodingsequences between the ORFs and at the genomic termini are shown in thepositive sense. From left to right, stop codons of indicated ORFs areshown, followed by the noncoding sequences, the gene start signals andstart codons of the indicated subsequent ORFs. Numbers indicate thefirst position of start and stop codons in the hMPV map. Sequences thatdisplay similarity to published gene end signals are underlined andsequences that display similarity to UAAAAAU/A/C (SEQ ID NO:172) arerepresented with a line above the sequence (SEQ ID NO:76, SEQ ID NO:77,SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82,SEQ ID NO:83, SEQ ID NO:84). (B) Nucleotide sequences of the genomictermini of Hmpv (SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88,SEQ ID NO:89, SEQ ID NO:90). The genomic termini of hMPV are alignedwith each other and with those of APV. Underlined regions represent theprimer sequences used in RT-PCR assays which are based on the 3′ and 5′end sequences of APV and RSV (Randhawa et al., 1997; Mink et al., 1991).Bold italicized nucleotides are part of the gene start signal of the Ngene. Le: leader, Tr: trailer.

FIG. 18: Comparison of two prototypic hMPV isolates with APV-A andAPV-C; DNA similarity matrices for nucleic acids encoding the variousviral proteins.

FIG. 19: Comparison of two prototypic hMPV isolates with APV-A andAPV-C; protein similarity matrices for the various viral proteins.

FIG. 20: Amino acid alignment of the nucleoprotein of two prototype hMPVisolates (SEQ ID NO:1, SEQ ID NO:91).

FIG. 21: Amino acid alignment of the phosphoprotein of two prototypehMPV isolates (SEQ ID NO:8, SEQ ID NO:92).

FIG. 22: Amino acid alignment of the matrix protein of two prototypehMPV isolates (SEQ ID NO:14, SEQ ID NO:93).

FIG. 23: Amino acid alignment of the fusion protein of two prototypehMPV isolates (SEQ ID NO:21, SEQ ID NO:94).

FIG. 24: Amino acid alignment of the M2-1 protein of two prototype hMPVisolates (SEQ ID NO:47, SEQ ID NO:95).

FIG. 25: Amino acid alignment of the M2-2 protein of two prototype hMPVisolates (SEQ ID NO:55, SEQ ID NO:96).

FIG. 26: Amino acid alignment of the short hydrophobic protein of twoprototype hMPV isolates (SEQ ID NO:63, SEQ ID NO:97).

FIG. 27: Amino acid alignment of the attachment glycoprotein of twoprototype hMPV isolates (SEQ ID NO:64, SEQ ID NO:98).

FIG. 28: Amino acid alignment of the N-terminus of the polymeraseprotein of two prototype hMPV isolates (SEQ ID NO:99, SEQ ID NO:100).

FIG. 29: Results of RT-PCR assays on throat and nose swabs of 12 guineapigs inoculated with ned/00/01 and/or ned/99/01.

FIG. 30A: IgG response against ned/00/01 and ned/99/01 for guinea pigsinfected with ned/00/01 and re-infected with ned/00/01 (GP 4, 5 and 6)or ned/99/01 (GP 1 and 3).

FIG. 30B: IgG response against ned/00/01 and ned/99/01 for guinea pigsinfected with ned/99/01 and re-infected with either ned/00/01 (GPs 8 and9) or with ned/99/01 (GPs 10, 11, 12).

FIG. 31: Specificity of the ned/00/01 and ned/99/01 ELISA on sera takenfrom guinea pigs infected with either ned/00/01 or ned/99/01.

FIG. 32: Mean IgG response against ned/00/01 and ned/99/01 ELISA of 3homologous (00-1/00-1), 2 homologous (99-1/99-1), 2 heterologous(99-1/00-1) and 2 heterologous (00-1/99-1) infected guinea pigs.

FIG. 33: Mean percentage of APV inhibition of hMPV-infected guinea pigs.

FIG. 34: Virus neutralization titers of ned/00/01 and ned/99/01 infectedguinea pigs against ned/00/01, ned/99/01 and APV-C.

FIG. 35: Results of RT-PCR assays on throat swabs of cynomolgus macaquesinoculated (twice) with ned/00/01.

FIG. 36 A (top two panels): IgA, IgM and IgG response against ned/00/01of 2 cynomolgus macaques (re)infected with ned/00/01.

FIG. 36B (bottom panels): IgG response against APV of 2 cynomolgusmacaques infected with ned/00/01.

FIG. 37: Comparison of the use of the hMPV ELISA and the APV inhibitionELISA for the detection of IgG antibodies in human sera.

DETAILED DESCRIPTION Virus Isolation and Characterization

From 1980 till 2000 we found 28 unidentified virus isolates frompatients with severe Respiratory disease. These 28 unidentified virusisolates grew slowly in tMK cells, poorly in VERO cells and A549 cellsand could not or only little be propagated in MDCK or chickenembryonated fibroblast cells. Most of these virus isolates induced CPEafter three passages on tMK cells, between day ten and fourteen. The CPEwas virtually indistinguishable from that caused by hRSV or hPIV in tMKor other cell cultures, characterized by syncytium formation after whichthe cells showed rapid internal disruption, followed by detachment ofthe cells from the monolayer. The cells usually (sometimes later)displayed CPE after three passages of virus from original material, atday 10 to 14 post inoculation, somewhat later than CPE caused by otherviruses such as hRSV or hPIV.

We used the supernatants of infected tMK cells for EM analysis whichrevealed the presence of paramyxovirus-like virus particles ranging from150 to 600 nanometers, with short envelope projections ranging from 13to 17 nanometers. Consistent with the biochemical properties ofenveloped viruses such as the Paramyxoviridae, standard chloroform orether treatment resulted in >10⁴ TCID50 reduction of infectivity for tMKcells. Virus-infected tMK cell culture supernatants did not displayhemagglutinating activity with turkey, chicken and guinea pigerythrocytes. During culture, the virus replication appeared to betrypsine dependent on the cells tested. These combined virological dataallowed that the newly identified virus was taxonomically classified asa member of the Paramyxoviridae family.

We isolated RNA from tMK cells infected with 15 of the unidentifiedvirus isolates for reverse transcription and polymerase chain reaction(RT-PCR) analyses using primer-sets specific for Paramyxovirinae,⁹ hPIV1-4, sendai virus, simian virus type 5, New-Castle disease virus, hRSV,morbilli, mumps, Nipah, Hendra, Tupaia and Mapuera viruses. RT-PCRassays were carried out at low stringency in order to detect potentiallyrelated viruses and RNA isolated from homologous virus stocks were usedas controls. Whereas the available controls reacted positive with therespective virus-specific primers, the newly identified virus isolatesdid not react with any primer set, indicating the virus was not closelyrelated to the viruses tested.

We used two of the virus-infected tMK cell culture supernatants toinoculate guinea pigs and ferrets intranasally. Sera were collected fromthese animals at day zero, two weeks and three weeks post inoculation.The animals displayed no clinical symptoms but all seroconverted asmeasured in virus neutralization (VN) assays and indirect IFA againstthe homologous viruses. The sera did not react in indirect IFA with anyof the known paramyxoviruses described above and with PVM. Next, wescreened the so far unidentified virus isolates using the guinea pig andferret pre- and post-infection sera, of which 28 were clearly positiveby indirect IFA with the post-infection sera suggesting they wereserological closely related or identical.

RAP PCR

To obtain sequence information on the unknown virus isolates, we used arandom PCR amplification strategy known as RAP-PCR.¹⁰ To this end, tMKcells were infected with one of the virus isolates (isolate 00-1) aswell as with hPIV-1 which served as a control. After both culturesdisplayed similar levels of CPE, virus in the culture supernatants waspurified on continuous 20-60% sucrose gradients. The gradient fractionswere inspected for virus-like particles by EM, and RNA was isolated fromthe fraction containing approximately 50% sucrose, in whichnucleocapsids were observed. Equivalent amounts of RNA isolated fromboth virus fractions were used for RAP-PCR, after which samples were runside by side on a 3% NuSieve agarose gel. Twenty differentiallydisplayed bands specific for the unidentified virus were subsequentlypurified from the gel, cloned in plasmid pCR2.1 (Invitrogen) andsequenced with vector-specific primers. When we used these sequences tosearch for homologies against sequences in the Genbank database usingthe BLAST software (www.ncbi.nlm.nih.gov/BLAST/) 10 out of 20 fragmentsdisplayed resemblance to APV/TRTV sequences.

These ten fragments were located in the genes coding for thenucleoprotein (N; fragments 1 and 2), the matrix protein (M; fragment3), the fusion protein A; fragments 4, 5, 6, 7) and the polymeraseprotein a; fragments 8, 9, 10) (FIG. 2). We next designed PCR primers tocomplete the sequence information for the 3′ end of the viral genomebased on our RAP PCR fragments as well as published leader and trailersequences for the Pneumovirinae.⁶ Three fragments were amplified, ofwhich fragment A spanned the extreme 3′ end of the N open reading frame(ORF), fragment B spanned the phosphoprotein (P) ORF and fragment Cclosed the gap between the M and F ORFs (FIG. 2). Sequence analyses ofthese three fragments revealed the absence of NS1 and NS2 ORFs at theextreme 3′ end of the viral genome and positioning of the F ORFimmediately adjacent to the M ORF. This genomic organization resemblesthat of the Metapneumovirus APV, which is also consistent with thesequence homology. Overall the translated sequences for the N, P, M andF ORFs showed an average of 30-33% homology with members of the genusPneumovirus and 66-68% with members of the genus Metapneumovirus. Forthe SH and G ORFS, no discernible homology was found with members ofeither of the genera. The amino acid homologies found for N showed about40% homology with hRSV and 88% with APV-C, its closest relativegenetically, as for example can be deduced by comparing the amino acidsequence of FIG. 3 with the amino acid sequence of the respective Nproteins of other viruses. The amino acid sequence for P showed about25% homology with hRSV and about 66-68% with APV-C, M showed about36-39% with hRSV and about 87-89% with APV-C, F showed about 40%homology with hRSV and about 81% with APV-C, M2-1 showed about 34-36%homology with Pneumoviruses and 84-86% with APV-C, M2-2 showed 15-17%homology with Pneumoviruses and 56% with APV-C and the fragmentsobtained in L showed an average of 44% with Pneumoviruses and 64% withAPV-C.

Phylogeny

Although BLAST searches using nucleotide sequences obtained from theunidentified virus isolate revealed homologies primarily with members ofthe Pneumovirinae, homologies based on protein sequences revealed someresemblance with other paramyxoviruses as well (data not shown). As anindication for the relation between the newly identified virus isolateand members of the Pneumovirinae, phylogenetic trees were constructedbased on the N, P, M and F ORFs of these viruses. In all fourphylogenetic trees, the newly identified virus isolate was most closelyrelated to APV (FIG. 4). From the four serotypes of APV that have beendescribed.¹¹ APV serotype C, the Metapneumovirus found primarily inbirds in the USA, showed the closest resemblance to the newly identifiedvirus. It should be noted however, that only partial sequenceinformation for APV serotype D is available.

To determine the relationship of our various newly identified virusisolates, we constructed phylogenetic trees based on sequenceinformation obtained from eight to nine isolates (8 for F, 9 for N, Mand L). To this end, we used RT-PCR with primers designed to amplifyshort fragments in the N, M, F and L ORFs, that were subsequentlysequenced directly. The nine virus isolates that were previously foundto be related in serological terms (see above) were also found to beclosely related genetically. In fact, all nine isolates were moreclosely related to one another than to APV. Although the sequenceinformation used for these phylogenetic trees was limited, it appearsthat the nine isolates can be divided in two groups, with isolate 94-1,99-1 and 99-2 clustering in one group and the other six isolates (94-2;93-1; 93-2; 93-3; 93-4; 00-1) in the other (FIG. 5).

Seroprevalence

To study the seroprevalence of this virus in the human population, wetested sera from humans in different age categories by indirect IFAusing tMK cells infected with one of the unidentified virus isolates.This analysis revealed that 25% of the children between six and twelvemonths had antibodies to the virus, and by the age of five nearly 100%of the children were seropositive. In total 56 serum samples tested byindirect IFA were tested by VN assay. For 51 (91%) of the samples theresults of the VN assay (titer >8) coincided with the results obtainedwith indirect IFA (titer>32). Four samples that were found positive inIFA, were negative by VN test (titer <8) whereas one serum reactednegative in IFA (titer<32) and positive in the VN test (titer 16) (table2).

IFA conducted with 72 sera taken from humans in 1958 (ages ranging from8-99 years)^(13, 27) revealed a 100% seroprevalence, indicating thevirus has been circulating in the human population for more than 40years. In addition a number of these sera were used in VN assays toconfirm the IFA data (table 2).

Genetic analyses of the N, M, P and F genes revealed that MPV has highersequence homology to the recently proposed genus Metapneumovirinae(average of 63%) as compared to the genus Pneumovirinae (average of 30%)and thus demonstrates a genomic organization similar to and resemblingthat of APV/TRTV. In contrast to the genomic organization of the RSVs(3-NS1-NS2-N-P-M-SH-G-F-M2-L-5′), Metapneumoviruses lack NS1 and NS2genes and have a different positioning of the genes between M and L(3-N-P-M-F-M2-SH-G-L-5′). The lack of ORFs between the M and F genes inour virus isolates and the lack of NS1 and NS2 adjacent to N, and thehigh amino acid sequence homology found with APV are reasons to proposethe classification of NPV isolated from humans as a first member of theMetapneumovirus genus of mammalian, in particular of human origin.

Phylogenetic analyses revealed that the nine MPV isolates from whichsequence information was obtained are closely related. Although sequenceinformation was limited, they were in fact more closely related to oneanother than to any of the avian Metapneumoviruses. Of the fourserotypes of APV that have been described, serotype C was most closelyrelated to MPV based on the N, P, M and F genes. It should be notedhowever that for serotype D only partial sequences for the F gene wereavailable from Genbank and for serotype B only M, N and F sequences wereavailable. Our MPV isolates formed two clusters in phylogenetic trees.For both hRSV and APV different genetic and serological subtypes havebeen described. Whether the two genetic clusters of MPV isolatesrepresent serological subgroups that are also functionally differentremains unknown at present. Our serological surveys showed that MPV is acommon human pathogen. The repeated isolation of this virus fromclinical samples from children with severe RTI indicates that theclinical and economic impact of MPV may be high. New diagnostic assaysbased on virus detection and serology will allow a more detailedanalysis of the incidence and clinical and economic impact of this viralpathogen.

The slight differences between the IFA and VN results (5 samples) maybedue to the fact that in the IFA only IgG serum antibodies were detectedwhereas the VN assay detects both classes and sub-classes of antibodiesor differences may be due to the differences in sensitivity between bothassays. For IFA a cut off value of 16 is used, whereas for VN a cut offvalue of 8 is used.

On the other hand, differences between IFA versus VN assay may alsoindicate possible differences between different serotypes of this newlyidentified virus. Since MPV seems most closely related to APV, wespeculate that the human virus may have originated from birds. Analysisof serum samples taken from humans in 1958 revealed that MPV has beenwidespread in the human population for more than 40 years indicatingthat a tentative zoonosis event must have taken place long before 1958.

Materials and Methods Specimen Collection

Over the past decades our laboratory has collected nasopharyngealaspirates from children suffering from RTI, which are routinely testedfor the presence of viruses. All nasopharyngeal aspirates were tested bydirect immunofluorescence assays (DIF) using fluorescence labeledantibodies against influenza virus types A, and B, hRSV and humanparainfluenza virus (hP) types 1 to 3. The nasopharyngeal aspirates werealso processed for virus isolation using rapid shell vial techniques¹⁴on various cell lines including VERO cells, tertiary cynomolgus monkeykidney (tMK) cells, human endothelial lung (HEL) cells and marbin dockkidney (MDCK) cells. Samples showing cytopathic effects (CPE) after twoto three passages, and which were negative in DIF, were tested byindirect immunofluorescence assays (IFA) using virus specific antibodiesagainst influenza virus types A, B and C, hRSV types A and B, measlesvirus, mumps virus, human parainfluenza virus (hPIV) types 1 to 4,sendai virus, simian virus type 5, and New-Castle disease virus.Although for many cases the etiological agent could be identified, somespecimens were negative for all these viruses tested.

Direct Immunofluorescence Assay (DIF)

Nasopharyngeal aspirate samples from patients suffering from RTI wereused for DIF and virus isolation as described.^(14, 15) Samples werestored at −70° C. In brief, nasopharyngeal aspirates were diluted with 5ml Dulbecco MEM (BioWhittaker, Walkersville, Md.) and thoroughly mixedon a vortex mixer for one minute. The suspension was thus centrifugedfor ten minutes at 840×g. The sediment was spread on a multispot slide(Nutacon, Leimuiden, The Netherlands), the supernatant was used forvirus isolation. After drying, the cells were fixed in aceton for 1minute at room temperature. After washing the slides were incubated for15 minutes at 37° C. with commercial available FITC-labeled virusspecific anti-sera such as influenza A and B, hRSV and hPIV 1 to 3(Dako, Glostrup, Denmark). After three washings in PBS and one in tapwater, the slides were included in a glycerol/PBS solution (Citifluor,UKC, Canterbury, UK) and covered. The slides were analyzed using aAxioscop fluorescence microscope (Carl Zeiss B. V, Weesp, theNetherlands.

Virus Isolation

For virus isolation tMK cells (RIVM, Bilthoven, The Netherlands) werecultured in 24-well plates containing glass slides (Costar, Cambridge,UK), with the medium described below supplemented with 10% fetal bovineserum (BioWhittaker, Vervier, Belgium). Before inoculation the plateswere washed with PBS and supplied with Eagle's MEM with Hanks' salt(ICN, Costa mesa, Calif.) of which half a liter was supplemented with0.26 gram HaHCO,³ 0.025 M Hepes (Biowhittaker), 2 mM L-glutamine(Biowhittaker), 100 units penicillin, 100 μg streptomycin(Biowhittaker), 0.5 gram lactal bumnine (Sigma-Aldrich, Zwijndrecht, TheNetherlands), 1.0 gram D-glucose (Merck, Amsterdam, The Netherlands),5.0 gram peptone (Oxoid, Haarlem, The Netherlands) and 0.02% trypsine(Life Technologies, Bethesda, Md.). The plates were inoculated withsupernatant of the nasopharyngeal aspirate samples, 0.2 ml per well intriplicate, followed by centrifuging at 840×g for one hour. Afterinoculation the plates were incubated at 37° C. for a maximum of 14 dayschanging the medium once a week and cultures were checked daily for CPE.After 14 days cells were scraped from the second passage and incubated14 days. This step was repeated for the third passage. The glass slideswere used to demonstrate the presence of the virus by indirect IFA asdescribed below.

Animal Immunization

Ferret and guinea pig specific antisera for the newly discovered viruswere generated by experimental intranasal infection of two specificpathogen free ferrets and two guinea pigs, housed in separatepressurized glove boxes. Two to three weeks later all the animals werebled by cardiac puncture, and their sera were used as reference sera.The sera were tested for all previous described viruses with indirectIFA as described below.

Antigen Detection by Indirect IFA

We performed indirect IFA on slides containing infected tMK cells. Afterwashing with PBS the slides were incubated for 30 minutes at 37° C. withvirus specific anti-sera. We used monoclonal antibodies in DIF againstinfluenza A, B and C, hPIV type 1 to 3 and hRSV as described above. ForhPIV type 4, mumps virus, measles virus, sendai virus, simian virus type5, New-Castle Disease virus polyclonal antibodies (RIVM) and ferret andguinea pig reference sera were used. After three washings with PBS andone wash with tap water, the slides were stained with a secondaryantibodies directed against the sera used in the first incubation.Secondary antibodies for the polyclonal anti sera were goat-anti-ferret(KPL, Guilford, UK, 40 fold diluted), mouse-anti-rabbit (Dako, Glostrup,Denmark, −20 fold diluted), rabbit-anti-chicken (KPL, 20 fold dilution)and mouse-anti-guinea pig (Dako, 20 fold diluted). Slides were processedas described for DIF.

Detection of Antibodies in Humans by Indirect IFA

For the detection of virus specific antibodies, infected tMK cells werefixed with cold acetone on coverslips, washed with PBS and stained withserum samples at a 1 to 16 dilution. Subsequently, samples were stainedwith FITC-labeled rabbit anti human antibodies 80 times diluted in PBS(Dako). Slides were processed as described above.

Virus Culture of MPV

Sub-confluent mono-layers of tMK cells in media as described above wereinoculated with supernatants of samples that displayed CPE after two orthree passages in the 24 well plates. Cultures were checked for CPEdaily and the media was changed once a week. Since CPE differed for eachisolate, all cultures were tested at day 12 to 14 with indirect IFAusing ferret antibodies against the new virus isolate. Positive cultureswere freeze-thawed three times, after which the supernatants wereclarified by low-speed centrifugation, aliquoted and stored frozen at−70° C. The 50% tissue culture infectious doses (TCID50) of virus in theculture supernatants were determined as described.¹⁶

Virus Neutralization Assay

VN assays were performed with serial two-fold dilutions of human andanimal sera starting at an eight-fold dilution. Diluted sera wereincubated for one hour with 100 TCID50 of virus before inoculation oftMK cells grown in 96 well plates, after which the plates werecentrifuged at 840×g. The media was changed after three and six days andIFA was conducted with ferret antibodies against MPV 8 days afterinoculation. The VN titer was defined as the lowest dilution of theserum sample resulting in negative IFA and inhibition of CPE in cellcultures.

Virus Characterization

Hemagglutination assays and chloroform sensitivity tests were performedas described.^(8, 14) For EM analyses, virus was concentrated frominfected cell culture supernatants in a micro-centrifuge at 4° C. at17000×g, after which the pellet was resuspended in PBS and inspected bynegative contrast EM. For RAP-PGR, virus was concentrated from infectedtMK cell supernatants by ultra-centrifugation on a 60% sucrose cushion(2 hours at 150000×g, 4° C.). The 60% sucrose interphase wassubsequently diluted with PBS and layered on top of a 20-60% continuoussucrose gradient which was centrifuged for 16 hours at 275000×g at 4° C.Sucrose gradient fractions were inspected for the presence of virus-likeparticles by EM and poly-acrylamide gel electrophoresis followed bysilver staining. The approximately 50% sucrose fractions that appearedto contain nucleocapsids were used for RNA isolation and RAP-PCR.

RNA Isolation

RNA was isolated from the supernatant of infected cell cultures orsucrose gradient fractions using a High Pure RNA Isolation kit accordingto instructions from the manufacturer (Roche Diagnostics, Almere, TheNetherlands).

RT-PCR

Virus-specific oligonucleotide sequences for RT-PCR assays on knownParamyxoviruses are described in addenda 1. A one-step RT-PCR wasperformed in 50 μl reactions containing 50 mM Tris.HCl pH 8.5, 50 mMNaCl, 4 mM MgCl₂, 2 mM dithiotreitol, 200 μM. each dNTP, 10 unitsrecombinant RNAsin (Promega, Leiden, the Netherlands), 10 units AMV RT(Promega, Leiden, The Netherlands), 5 units Amplitaq Gold DNA polymerase(PE Biosystems, Nieuwerkerk aan de Ijssel The Netherlands) and 5 μl RNA.Cycling conditions were 45 min. at 42° C. and 7 min. at 95° C. once, 1min at 95° C., 2 min. at 42° C. and 3 min. at 72° C. repeated 40 timesand 10 min. at 72° C. once.

RAP-PCR

RAP-PCR was performed essentially as described.¹⁰ The oligonucleotidesequences are described in addenda 2. For the RT reaction, 2 μl RNA wasused in a 10 μl reaction containing 10 ng/μl oligonucleotide, 10 mMdithiotreitol, 500 μm each dNTP, 25 mM Tris-HCl pH 8.3, 75 mM KCl and 3mM MgCl₂. The reaction mixture was incubated for 5 min. at 70° C. and 5min. at 37° C., after which 200 units Superscript RT enzyme(LifeTechnologies) were added. The incubation at 37° C. was continuedfor 55 min. and the reaction terminated by a 5 min. incubation at 72° C.The RT mixture was diluted to give a 50 μl PCR reaction containing 8ng/μl oligonucleotide, 300 μm each dNTP, 15 mM Tris-HCL pH 8.3, 65 mMKCl, 3.0 mM MgCl₄ and 5 units Taq DNA polymerase (PE Biosystems).Cycling conditions were 6 mM. at 94° C., 5 min. at 40° C. and 1 min. at72° C. once, followed by 1 min. at 94° C., 2 min. at 56° C. and 1 min.at 72° C. repeated 40 times and 6 min. at 72° C. once. After RAP-PCR, 15μl the RT-PCR products were run side by side on a 3% NuSieve agarose gel(FMC BioProducts, Heerhugowaard, The Netherlands). Differentiallydisplayed fragments specific for MPV were purified from the gel withQiaquick Gel Extraction kit (Qiagen, Leusden, The Netherlands) andcloned in pCR2.1 vector (Invitrogen, Groningen, The Netherlands)according to instructions from the manufacturer.

Sequence Analysis

RAP-PCR products cloned in vector pCR2.1 (Invitrogen) were sequencedwith M13-specific oligonucleotides. DNA fragments obtained by RT-PCRwere purified from agarose gels using Qiaquick Gel Extraction kit(Qiagen, Leusden, The Netherlands), and sequenced directly with the sameoligonucleotides used for PCR. Sequence analyses were performed using aDyenamic ET terminator sequencing kit (Amersham Pharmacia Biotech,Roosendaal, The Netherlands) and an ABI 373 automatic DNA sequencer (PEBiosystem). All techniques were performed according to the instructionsof the manufacturer.

Generating Genomic Fragments of MPV by RT-PCR

To generate PCR fragments spanning gaps A, B and C between the RAP-PCRfragments (FIG. 2) we used RT-PCR assays as described before on RNAisolated from virus isolate 00-1. The following primers were used:

-   -   For fragment A: TR1 designed in the leader:        (5′-AAAGAATTCACGAGAAAAAAACGC-3′) (SEQ ID NO:107) and N1 designed        at the 3′ end of the RAP-PCR fragments obtained in N        (5′-CTGTGGTCTCTAGTCCCACTTC-3′) (SEQ ID NO:108).    -   For fragment B: N2 designed at the 5′ end of the RAP-PCR        fragments obtained in N: (5′-CATGCAAGCTTATGGGGC-3′) (SEQ ID        NO:19) and M1 designed at the 3′ end of the RAP-PCR fragments        obtained in M: (5′-CAGAGTGGTTATTGTCAGGGT-3) (SEQ ID NO:110).    -   For fragment C: M2 designed at the 5′ end of the RAP-PCR        fragment obtained in M: (5′-GTAGAACTAGGAGCATATG-3′) (SEQ ID        NO:111) and F1 designed at the 3′ end of the RAP-PCR fragments        obtained in F: (5′-TCCCCAATGTAGATACTGCTTC-3′) (SEQ ID NO:112).

Fragments were purified from the gel, cloned and sequenced as describedbefore.

RT-PCR for Diagnosing MPV

For the amplification and sequencing of parts of the N, M, F and L ORFsof nine of the MPV isolates, we used primers N3(5′-GCACTCAAGAGATACCCTAG-3′) (SEQ ID NO:113) and N4(5′-AGACTTTCTGCTTTGCTGCCTG-3′) (SEQ ID NO:114), amplifying a 151nucleotide fragments, M3 (5′-CCCTGACAATAACCACTCTG-3′) (SEQ ID NO:115)and M4 (5′-GCCAACTGATTTGGCTGAGCTC-3′) (SEQ ID NO:116) amplifying a 252nucleotide fragment, F7 (5′-TGCACTATCTCCTCTTGGGGCTTTG-3′) (SEQ IDNO:117) and F8 (5′-TCAAAGCTGCTTGACACTGGCC-3′) (SEQ ID NO:118) amplifyinga 221 nucleotide fragment and L6 (5′-CATGCCCACTATAAAAGGTCAG-3′) (SEQ IDNO:119) and L7 (5′-CACCCCAGTCTTTCTTGAAA-3′) (SEQ ID NO:120) amplifying a173 nucleotide fragment respectively. RT-PCR, gel purification anddirect sequencing were performed as described above. Furthermore, probesused were:

Probe used in M: (SEQ ID NO: 121) 5′-TGC TTG TAC TTC CCA AAG-3′Probe used in N: (SEQ ID NO: 122) 5′-TAT TTG AAC AAA AAG TGT-3′Probe used in L: (SEQ ID NO: 123) 5′-TGGTGTGGGATATTAACAG-3′

Phylogenetic Analyses

For all phylogenetic trees, DNA sequences were aligned using theClustalW software package and maximum likelihood trees were generatedusing the DNA-ML software package of the Phylip 3.5 program using 100bootstraps and 3 jumbles.¹⁵ Previously published sequences that wereused for the generation of phylogenetic trees are available from Genbankunder accessions numbers: For all ORFs: hRSV: NC001781; bRSV: NC001989;For the F ORF: PVM, D11128; APV-A, D00850; APV-B, Y14292; APV-C,AF187152; For the N ORF: PVM, D10331; APV-A, U39295; APV-B, U39296;APV-C, AF176590; For the M ORF: PMV, U66893; APV-A, X58639; APV-B,U37586; APV-C, AF262571; For the P ORF: PVM, 09649; APV-A, U22110,APV-C, AF176591. Phylogenetic analyses for the nine different virusisolates of MPV were performed with APV strain C as outgroup.

Abbreviations used in figures: hRSV: human RSV; bRSV: bovine RSV; PVM:pneumonia virus of mice; APV-A, B, and C: avian Pneumovirus type A, Band C.

Examples of Methods to Identify MPV Specimen Collection

In order to find virus isolates nasopharyngeal aspirates, throat andnasal swabs, bronchoalveolar lavages preferably from mammals such ashumans, carnivores (dogs, cats, mustelids, seals etc.), horses,ruminants (cattle, sheep, goats etc.), pigs, rabbits, birds (poultry,ostriches, etc.) should be examined. From birds cloaca swabs anddroppings can be examined as well Sera should be collected forimmunological assays, such as ELISA and virus neutralization assays.

Collected virus specimens were diluted with 5 ml Dulbecco MEM medium(BioWhittaker, Walkersville, Md.) and thoroughly mixed on a vortex mixerfor one minute. The suspension was thus centrifuged for ten minutes at840×g. The sediment was spread on a multispot slide (Nutacon, Leimuiden,The Netherlands) for immunofluorescence techniques, and the supernatantwas used for virus isolation.

Virus Isolation

For virus isolation tMK cells (RIVM, Bilthoven, The Netherlands) werecultured in 24 well plates containing glass slides (Costar, Cambridge,UK, with the medium described below supplemented with 10% fetal bovineserum (BioWhittaker, Vervier, Belgium). Before inoculation the plateswere washed with PBS and supplied with Eagle's MEM with Hanks' salt(ICN, Costa Mesa, Calif.) supplemented with 0.52/liter gram NaHCO₃,0.025 M Hepes (Biowhittaker), 2 mM Iglutamine (Biowhittaker), 200units/liter penicillin, 200 μg/liter streptomycin (Biowhittaker), 1gram/liter lactalbumin (Sigma-Aldiich, Zwindrecht, The Netherlands), 2.0gram/liter D-glucose (Merck, Amsterdam, The Netherlands), 10 gram/literpeptone (Oxoid, Haarlem, The Netherlands) and 0.02% trypsine (LifeTechnologies, Bethesda, Md.).

The plates were inoculated with supernatant of the nasopharyngealaspirate samples, 0.2 ml per well in triplicate, followed bycentrifuging at 840×g for one hour. After inoculation the plates wereincubated at 37° C. for a maximum of 14 days changing the medium once aweek and cultures were checked daily for CPE. After 14 days, cells werescraped from the second passage and incubated for another 14 days. Thisstep was repeated for the third passage. The glass slides were used todemonstrate the presence of the virus by indirect IFA as describedbelow.

CPE was generally observed after the third passage, at day 8 to 14depending on the isolate. The CPE was virtually indistinguishable fromthat caused by hRSV or hPIV in tMK or other cell cultures. However, hRSVinduces CPE starting around day 4. CPE was characterized by syncytiaformation, after which the cells showed rapid internal disruption,followed by detachment of cells from the monolayer. For some isolatesCPE was difficult to observe, and IFA was used to confirm the presenceof the virus in these cultures.

Virus Culture of MPV

Sub-confluent monolayers of tMK cells in media as described above wereinoculated with supernatants of samples that displayed CPE after two orthree passages in the 24 well plates. Cultures were checked for CPEdaily and the media was changed once a week. Since CPE differed for eachisolate, all cultures were tested at day 12 to 14 with indirect IFAusing ferret antibodies against the new virus isolate. Positive cultureswere freeze-thawed three times, after which the supernatants wereclarified by low-speed centrifugation, aliquoted and stored frozen at−70° C. The 50% tissue culture infectious doses (TCID50) of virus in theculture supernatants were determined following established techniquesused in the field.¹⁶

Virus Characterization

Hemagglutination assays and chloroform sensitivity tests were performedfollowing well established and described techniques used in the field.¹⁴For EM analyses, virus was concentrated from infected cell culturesupernatants in a micro-centrifuge at 4° C. at 17000×g, after which thepellet was resuspended in PBS and inspected by negative contrast EM.

Antigen Detection by Indirect IFA

Collected specimens were processed as described and sediment of thesamples was spread on a multispot slide. After drying, the cells werefixed in aceton for 1 minute at room temperature.

Alternatively, virus was cultured on tMK cells in 24 well slidescontaining glass slides. These glass slides were washed with PBS andfixed in aceton for 1 minute at room temperature.

After washing with PBS the slides were incubated for 30 minutes at 37°C. with polyclonal antibodies at a dilution of 1:50 to 1:100 in PBS. Weused immunized ferrets and guinea pigs to obtain polyclonal antibodies,but these antibodies can be raised in various animals, and the workingdilution of the polyclonal antibody can vary for each immunization.After three washes with PBS and one wash with tap water, the slides wereincubated at 37° C. for 30 minutes with FITC labeled goat-anti-ferretantibodies (KPL, Guilford, UK, 40 fold diluted). After three washes inPBS and one in tap water, the slides were included in a glycerol/PBSsolution (Citifluor, UKC, Canterbury, UK) and covered. The slides wereanalyzed using an Axioscop fluorescence microscope (Carl Zeiss B. V.,Weesp, the Netherlands). Detection of antibodies in humans, mammals,ruminants or other animals by indirect.

IFA

For the detection of virus specific antibodies, infected tMK cells withMPV were fixed with acetone on coverslips (as described above), washedwith PBS and incubated 30 minutes at 37° C. with serum samples at a 1 to16 dilution. After two washes with PBS and one with tap water, theslides were incubated 30 minutes at 37° C. with FITC-labeled secondaryantibodies to the species used (Dako). Slides were processed asdescribed above.

Antibodies can be labeled directly with a fluorescent dye, which willresult in a direct immuno fluorescence assay. FITC can be replaced withany fluorescent dye.

Animal Immunization

Ferret and guinea pig specific antisera for the newly discovered viruswere generated by experimental intranasal infection of two specificpathogen free ferrets and two guinea pigs, housed in separatepressurized glove boxes. Two to three weeks later the animals were bledby cardiac puncture, and their sera were used as reference sera.

The sera were tested for all previous described viruses with indirectIFA as described below. Other animal species are also suitable for thegeneration of specific antibody preparations and other antigenpreparations may be used.

Virus Neutralization Assay (VN Assay)

VN assays were performed with serial two-fold dilutions of human andanimal sera starting at an eight-fold dilution. Diluted sera wereincubated for one hour with 100 TCID50 of virus before inoculation oftMK cells grown in 96 well plates, after which the plates werecentrifuged at 840×g. The same culture media as described above wasused. The media was changed after three and six days, and after 8 daysIFA was performed (see above). The VN titer was defined as the lowestdilution of the serum sample resulting in negative IFA and inhibition ofCPE in cell cultures.

RNA Isolation

RNA was isolated from the supernatant of infected cell cultures orsucrose gradient fractions using a High Pure RNA Isolation kit accordingto instructions from the manufacturer (Roche Diagnostics, Almere, TheNetherlands). RNA can also be isolated following other procedures knownin the field (Current Protocols in Molecular Biology).

RT-PCR

A one-step RT-PCR was performed in 50 μl reactions containing 50 mMTris.HCl pH 8.5, 50 mM NaCl, 4 mM MgCl₂, 2 mM dithiotreitol, 200 μM eachdNTP, 10 units recombinant RNAsin (Promega, Leiden, the Netherlands), 10units AMV RT (Promega, Leiden, The Netherlands), 5 units Amplitaq GoldDNA polymerase (PE Biosystems, Nieuwerkerk aan de Ijssel, TheNetherlands) and 5 μl RNA. Cycling conditions were 45 min. at 42° C. and7 min. at 95° C. once, 1 min at 95° C., 2 min. at 42° C. and 3 min. at72° C. repeated 40 times and 10 min. at 72° C. once.

Primers used for diagnostic PCR:

-   -   In the nucleoprotein: N3 (5′-GCACTCAAGAGATACCCTAG-3′) (SEQ ID        NO:124) and N4 (5′-AGACTTTCTGCTTTGCTGCCTG-3′) (SEQ ID NO:125),        amplifying a 151 nucleotide fragment.    -   In the matrix protein: M3 (5′-CCCTGACAATAACCACTCTG-3′) (SEQ ID        NO:126) and M4 (5′-GCCAACTGATTTGGCTGAGCTC-3′) (SEQ ID NO:127)        amplifying a 252 nucleotide fragment.    -   In the polymerase protein: L6 (5′-CATGCCCACTATAAAAGGTCAG-3′)        (SEQ ID NO:128) and L7 (5′-CACCCCAGTCTTTCTTGAAA-3′) (SEQ ID        NO:129) amplifying a 173 nucleotide fragment.

Other primers can be designed based on MPV sequences, and differentbuffers and assay conditions may be used for specific purposes.

Sequence Analysis

Sequence analyses were performed using a Dyenamic ET terminatorsequencing kit (Amersham Pharmacia Biotech, Roosendaal, The Netherlands)and an ABI 373 automatic DNA sequencer (PE Biosystem). All techniqueswere performed according to the instructions of the manufacturer. PCRfragments were sequenced directly with the same oligonucleotides usedfor PCR, or the fragments were purified from the gel with Qiaquick GelExtraction kit (Qiagen, Leusden, The Netherlands) and cloned in pCR2.1vector (Invitrogen, Groningen, The Netherlands) according toinstructions from the manufacturer and subsequently sequenced withM13-specific oligonucleotides.

Oligonucleotides Used for Analyzing the 3′ End of the Genome (Absence ofNS1/NS2).

Primer TR1 (5′-AAAGAATTCACGAGAAAAAAACGC-3′) (SEQ ID NO:130) was designedbased on published sequences of the trailer and leader for hRSV and APV,published by Randhawa (1997) and primer N1(5′-CTGTGGTCTCTAGTCCCACTTC-3′) (SEQ ID NO:131) was designed based onobtained sequences in the N protein. The RT-PCR assay and sequencing wasperformed as described above.

The RT-PCR gave a product of approximately 500 base pairs which is toosmall to contain information for two ORFS, and translation of thesesequences did not reveal an ORF. Detection of Antibodies in Humans,Mammals, Ruminants or Other Animals by ELISA

In Paramyxoviridae, the N protein is the most abundant protein, and theimmune response to this protein occurs early in infection. For thesereasons, a recombinant source of the N proteins is preferably used fordeveloping an ELISA assay for detection of antibodies to MPV. Antigenssuitable for antibody detection include any MPV protein that combineswith any MPV-specific antibody of a patient exposed to or infected withMPV virus. Preferred antigens of the invention include those thatpredominantly engender the immune response in patients exposed to MPV,which therefore, typically are recognized most readily by antibodies ofa patient. Particularly preferred antigens include the N, F and Gproteins of MPV. Antigens used for immunological techniques can benative antigens or can be modified versions thereof. Well knowntechniques of molecular biology can be used to alter the amino acidsequence of a MPV antigen to produce modified versions of the antigenthat may be used in immunologic techniques.

Methods for cloning genes, for manipulating the genes to and fromexpression vectors, and for expressing the protein encoded by the genein a heterologous host are well-known, and these techniques can be usedto provide the expression vectors, host cells, and the for expressingcloned genes encoding antigens in a host to produce recombinant antigensfor use in diagnostic assays. See for instance: Molecular cloning, Alaboratory manual and Current Protocols in Molecular Biology.

A variety of expression systems may be used to produce MPV antigens. Forinstance, a variety of expression vectors suitable to produce proteinsin E. Coli, B. subtilis, yeast, insect cells and mammalian cells havebeen described, any of which might be used to produce a MPV antigensuitable to detect anti-MPV antibodies in exposed patients.

The baculovirus expression system has the advantage of providingnecessary processing of proteins, and is therefore preferred. The systemutilizes the polyhedrin promoter to direct expression of MPV antigens.(Matsuura et al. 1987, J. Gen. Virol. 68:1233-1250).

Antigens produced by recombinant baculo-viruses can be used in a varietyof immunological assays to detect anti-MPV antibodies in a patient. Itis well established, that recombinant antigens can be used in place ofnatural virus in practically any immunological assay for detection ofvirus specific antibodies. The assays include direct and indirectassays, sandwich assays, solid phase assays such as those using platesor beads among others, and liquid phase assays. Assays suitable includethose that use primary and secondary antibodies, and those that useantibody binding reagents such as protein A. Moreover, a variety ofdetection methods can be used in the invention, including calorimetric,fluorescent, phosphorescent, chemiluminescent, luminescent andradioactive methods.

Example 1 Of Indirect Anti-MPV IgG EIA Using Recombinant N Protein

An indirect IgG EIA using a recombinant N protein (produced withrecombinant baculo-virus in insect (Sf9) cells) as antigen can beperformed. For antigen preparation, Sf9 cells are infected with therecombinant baculovirus and harvested 3-7 days post infection. The cellsuspension is washed twice in PBS, pH 7.2, adjusted to a cell density of5.0×10⁶ cells/ml, and freeze-thawed three times. Large cellular debrisis pelleted by low speed centrifugation (500×g for 15 min.) and thesupernatant is collected and stored at −70° C. until use. Uninfectedcells are processed similarly for negative control antigen.

100 μl of a freeze-thaw lysate is used to coat microtiter plates, atdilutions ranging from 1:50 to 1:1000. An uninfected cell lysate is runin duplicate wells and serves as a negative control. After incubationovernight, plates are washed twice with PBS/0.05% TWEEN®. Test sera arediluted 1:50 to 1:200 in ELISA buffer (PBS, supplemented to 2% withnormal goat sera, and with 0.5% bovine serum albumin and 0.1% milk),followed by incubation wells for 1 hour at 37° C.

Plates are washed two times with PBS/0.05% TWEEN®. Horseradishperoxidase labeled goat anti-human (or against other species) IgG,diluted 1:3000 to 1:5000 in ELISA buffer, added to wells, and incubatedfor 1 hour at 37° C. The plates are then washed two times with PBS/0.05%TWEEN® and once with tap water, incubated for 15 minutes at roomtemperature with the enzyme substrate TMB, 3, 3′, 5, 5′tetramethylbenzidine, such as that obtained from Sigma, and the reactionis stopped with 100 μl of 2 M phosphoric acid. Colorimetric readings aremeasured at 450 nm using an automated microtiter plate reader.

Example 2 Capture Anti-MPV IgM EIA Using a Recombinant Nucleoprotein

A capture IgM EIA using the recombinant nucleoprotein or any otherrecombinant protein as antigen can be performed by modification ofassays as previously described by Erdman et al. (1990), J. Clin. Microb.29:1466-1471.

Affinity purified anti-human IgM capture antibody (or against otherspecies), such as that obtained from Dako, is added to wells of amicrotiter plate in a concentration of 250 ng per well in 0.1 Mcarbonate buffer pH 9.6. After overnight incubation at room temperature,the plates are washed two times with PBS/0.05% TWEEN®. 100 μl of testserum diluted 1:200 to 1:1000 in ELISA buffer is added to triplicatewells and incubated for 1 hour at 37° C. The plates are then washed twotimes with in PBS/0.05% TWEEN®.

The freeze-thawed (infected with recombinant virus) Sf21 cell lysate isdiluted 1:100 to 1:500 in ELISA buffer is added to the wells andincubated for 2 hours at 37° C. Uninfected cell lysate serves as anegative control and is run in duplicate wells.

The plates are then washed three times in PBS/0.05% TWEEN® and incubatedfor 1 hour at 37° C. with 100 μl of a polyclonal antibody against MPV inan optimal dilution in ELISA buffer. After two washes with PBS/0.05%TWEEN®, the plates are incubated with horseradish peroxide labeledsecondary antibody (such as rabbit anti ferret), and the plates areincubated 20 minutes at 37° C.

The plates are then washed five times in PBS/0/05% TWEEN®, incubated for15 minutes at room temperature with the enzyme substrate TMB 3, 3′, 5,5′ tetramethylbenzidine, as, for instance obtained from Sigma, and thereaction is stopped with 100 μl of 2 M phosphoric acid. Colorimetricreadings are measured at 450 nm using automated microtiter plate reader.

The sensitivities of the capture IgM EIAs using the recombinantnucleoprotein (or other recombinant protein) and whole MPV virus arecompared using acute-and convalescent-phase serum pairs form personswith clinical MPV virus infection. The specificity of the recombinantnucleoprotein capture EIA is determined by testing serum specimens fromhealthy persons and persons with other paramyxovirus infections.

Potential for ELAs for using recombinant MPV fusion and glycoproteinproteins produced by the baculovirus expression.

The glycoproteins G and F are the two transmembraneous envelopeglycoproteins of the MPV virion and represent the major neutralizationand protective antigens. The expression of these glycoproteins in avector virus system such as a baculovirus system provides a source ofrecombinant antigens for use in assays for detection of MPV specificantibodies. Moreover, their use in combination with the nucleoprotein,for instance, further enhances the sensitivity of enzyme immunoassays inthe detection of antibodies against MPV.

A variety of other immunological assays (Current Protocols inImmunology) may be used as alternative methods to those described here.

In order to find virus isolates nasopharyngeal aspirates, throat andnasal swabs, bronchoalveolar lavages and throat swabs preferable frombut not limited to humans, carnivores (dogs, cats, seals, etc.), horses,ruminants (cattle, sheep, goats, etc.), pigs, rabbits, birds (poultry,ostriches, etc.) can be examined. From birds, cloaca and intestinalswabs and droppings can be examined as well. For all samples, serology(antibody and antigen detection, etc.), virus isolation and nucleic aciddetection techniques can be performed for the detection of virus.Monoclonal antibodies can be generated by immunizing mice (or otheranimals) with purified MPV or parts thereof (proteins, peptides) andsubsequently using established hybridoma technology (Current Protocolsin Immunology). Alternatively, phage display technology can be used forthis purpose (Current Protocols in Immunology). Similarly, polyclonalantibodies can be obtained from infected humans or animals, or fromimmunized humans or animals (Current Protocols in Immunology).

The detection of the presence or absence of NS1 and NS2 proteins can beperformed using Western blotting, IFA, immuno precipitation techniquesusing a variety of antibody preparations. The detection of the presenceor absence of NS1 and NS2 genes or homologues thereof in virus isolatescan be performed using PCR with primer sets designed on the basis ofknown NS1 and/or NS2 genes as well as with a variety of nucleic acidhybridization techniques.

To determine whether NS1 and NS2 genes are present at the 3′ end of theviral genome, a PCR can be performed with primers specific for this 3′end of the genome. In our case, we used a primer specific for the 3′untranslated region of the viral genome and a primer in the N ORF. Otherprimers may be designed for the same purpose. The absence of the NS1/NS2genes is revealed by the length and/or nucleotide sequence of the PCRproduct. Primers specific for NS1 and/or NS2 genes may be used incombination with primers specific for other parts of the 3′ end of theviral genome (such as the untranslated region or N, M or F ORFs) toallow a positive identification of the presence of NS1 or NS2 genes. Inaddition to PCR, a variety of techniques such as molecular cloning,nucleic acid hybridization may be used for the same purpose.

Example 3 Different Serotypes/Subgroups of MPV

Two potential genetic clusters are identified by analyses of partialnucleotide sequences in the N, M, F and L ORFs of 9 virus isolates.90-100% nucleotide identity was observed within a cluster, and 81-88%identity was observed between the clusters. Sequence informationobtained on more virus isolates confirmed the existence of twogenotypes. Virus isolate ned/00/01 as prototype of cluster A, and virusisolate ned/99/01 as prototype of cluster B have been used incross-neutralization assays to test whether the genotypes are related todifferent serotypes or subgroups.

Results

Using RT-PCR assays with primers located in the polymerase gene, weidentified 30 additional virus isolates from nasopharyngeal aspiratesamples. Sequence information of parts of the matrix and polymerasegenes of these new isolates together with those of the previous 9isolates were used to construct phylogenetic trees (FIG. 16). Analysesof these trees confirmed the presence of two genetic clusters, withvirus isolate ned/00/00-1 as the prototype virus in group A and virusisolate ned/99/01 as the prototype virus in group B. The nucleotidesequence identity within a group was more than 92%, while between theclusters the identity was 81-85%.

Virus isolates ned/00/01 and ned/99/01 have been used to inoculateferrets to raise virus-specific antisera. These antisera were used invirus neutralization assays with both viruses.

TABLE 3 Virus neutralization titers isolate 00-1 isolate 99-1 preserumferret A (00-1) .2 .2 ferret A 22 dpi (00-1) 64 .2 preserum ferret B(99-1) .2 .2 ferret B 22 dpi (99-1) 4 64

For isolate 00-1 the titer differs 32 (64/2) fold

For isolate 99-1 the titer differs 16 (64/4) fold

In addition, 6 guinea pigs have been inoculated with either one of theviruses (ned/00/01 and ned/99/01). RT-PCR assays on nasopharyngealaspirate samples showed virus replication from day 2 till day 10 postinfection. At day 70 post infection the guinea pigs have been challengedwith either the homologous or the heterologous virus, and for in allfour cases virus replication has been noticed.

TABLE 4 primary virus secondary virus infection replication infectionreplication guinea pig 1-3 00-1 2 out of 3 99-1 1 out of 2 guinea pig4-6 00-1 3 out of 3 00-1 1 out of 3 guinea pig 7-9 99-1 3 out of 3 00-12 out of 2 guinea pig 10-12 99-1 3 out of 3 99-1 1 out of 3 note: forthe secondary infection guinea pig 2 and 9 were not there anymore.

Virus neutralization assays with antisera after the first challengeshowed essentially the same results as in the VN assays performed withthe ferrets (>16-fold difference in VN titer).

The results presented in this example confirm the existence of twogenotypes, which correspond to two serotypes of MPV, and show thepossibility of repeated infection with heterologous and homologous virus

Example 4 Further Sequence Determination

This example describes the further analysis of the sequences of MPV openreading frames (ORFs) and intergenic sequences as well as partialsequences of the genomic termini.

Sequence analyses of the nucleoprotein (N), phosphoprotein (P), matrixprotein (M) and fusion protein (F) genes of MPV revealed the highestdegree of sequence homology with APV serotype C, the avian Pneumovirusfound primarily in birds in the United States. These analyses alsorevealed the absence of non-structural proteins NS1 and NS2 at the 3′end of the viral genome and positioning of the fusion proteinimmediately adjacent to the matrix protein. Here we present thesequences of the 22K (M2) protein, the small hydrophobic (SH) protein,the attachment (G) protein and the polymerase (L) protein genes, theintergenic regions and the trailer sequence. In combination with thesequences described previously the sequences presented here complete thegenomic sequence of MPV with the exception of the extreme 12-15nucleotides of the genomic termini and establish the genomicorganization of MPV. Side by side comparisons of the sequences of theMPV genome with those of APV subtype A, B and C, RSV subtype A and B,PVM and other paramyxoviruses provides strong evidence for theclassification of MPV in the Metapneumovirus genus.

Results Sequence Strategy

MTV isolate 00-1 (van den Hoogen et al., 2001) was propagated intertiary monkey kidney (tMK) cells and RNA isolated from the supernatant3 weeks after inoculation was used as template for RT-PCR analyses.Primers were designed on the basis of the partial sequence informationavailable for MPV 00-1 (van den Hoogen et al., 2001) as well as theleader and trailer sequences of APV and RSV (Randhawa et al., 1997; Minket al., 1991). Initially, fragments between the previously obtainedproducts, ranging in size from 500 bp to 4 Kb in length, were generatedby RT-PCR amplification and sequenced directly. The genomic sequence wassubsequently confirmed by generating a series of overlapping RT-PCRfragments ranging in size from 500 to 800 bp that represented the entireMPV genome. For all PCR fragments, both strands were sequenced directlyto minimize amplification and sequencing errors. The nucleotide andamino acid sequences were used to search for homologies with sequencesin the Genbank database using the BLAST software (on the Internet atncbi.nlm.nih.gov/BLAST). Protein names were assigned to open readingframes (ORFs) based on homology with known viral genes as well as theirlocation in the genome. Based on this information, a genomic map for MPVwas constructed (FIG. 7). The MPV genome is 13378 nucleotides in lengthand its organization is similar to the genomic organization of APV.Below, we present a comparison between the ORFs and non-coding sequencesof MPV and those of other paramyxoviruses and discuss the importantsimilarities and differences.

The Nucleoprotein (N) Gene

As shown, the first gene in the genomic map of MPV codes for a 394 aminoacid (aa) protein and shows extensive homology with the N protein ofother Pneumoviruses. The length of the N ORF is identical to the lengthof the N ORF of APV-C (Table 5) and is smaller than those of otherparamyxoviruses (Barr et al., 1991). Analysis of the amino acid sequencerevealed the highest homology with APV-C (88%), and only 7-11% withother paramyxoviruses (Table 6).

Barr et al. (1991) identified three regions of similarity betweenviruses belonging to the order Mononegavirales: A, B and C (FIG. 8).Although similarities are highest within a virus family, these regionsare highly conserved between virus families. In all three regions MPVrevealed 97% aa sequence identity with APV-C, 89% with APV-B, 92% withAPV-A, and 66-73% with RSV and PVM. The region between aa residues 160and 340 appears to be highly conserved among Metapneumoviruses and to asomewhat lesser extent the Pneumovirinae (Miyahara et al., 1992; Li etal., 1996; Barr et al., 1991). This is in agreement with MPV being aMetapneumovirus, showing 100% similarity with APV C.

Th Phosphoprotein (P) Gene

The second ORF in the genome map codes for a 294 aa protein which shares68% aa sequence homology with the P protein of APV-C, and only 22-26%with the P protein of RSV (Table 6). The P gene of MPV contains onesubstantial ORF and in that respect is similar to P from many otherparamyxoviruses (reviewed in Lamb and Kolakofsky, 1996; Sedlieier etal., 1998).

In contrast to APV A and B and PVM and similar to RSV and APV-C the MPVP ORF lacks cysteine residues. Ling (1995) suggested that a region ofhigh similarity between all Pneumoviruses (aa 185-241) plays a role ineither the RNA synthesis process or in maintaining the structuralintegrity of the nucleocapsid complex. This region of high similarity isalso found in MPV (FIG. 9) especially when conservative substitutionsare taken in account, showing 100% similarity with APV-C, 93% with APV-Aand B, and approximately 81% with RSV. The C-terminus of the MPV Pprotein is rich in glutamate residues as has been described for APVs(Ling et al., 1995).

The Matrix (M) Protein Gene

The third ORF of the MPV genome encodes a 254 aa protein, whichresembles the M ORFs of other Pneumoviruses. The M ORF of MPV hasexactly the same size as the M ORFs of other Metapneumoviruses (Table 5)and shows high aa sequence homology with the matrix proteins of APV(78-87%), lower homology with those of RSV and PVM (37-38%) and 10% orless homology with those of other paramyxoviruses (Table 6).

Easton (1997) compared the sequences of matrix proteins of allPneumoviruses and found a conserved heptapeptide at residue 14 to 19that is also conserved in MPV (FIG. 10). For RSV, PVM and APV smallsecondary ORFs within or overlapping with the major ORF of M have beenidentified (52 aa and 51 aa in bRSV, 76 aa in RSV, 46 aa in PVM and 51aa in APV) (Yu et al., 1992; Easton et al., 1997; Samal et al., 1991;Satake et al., 1984). We noticed two small ORFs in the M ORF of MPV. Onesmall ORF of 54 aa residues was found within the major M ORF (fragment1, FIG. 7), starting at nucleotide 2281 and one small ORF of 33 aaresidues was found overlapping with the major ORF of M starting atnucleotide 2893 (fragment 2, FIG. 7). Similar to the secondary ORFs ofRSV and APV there is no significant homology between these secondaryORFs and secondary ORFs of the other Pneumoviruses, and apparent startor stop signals are lacking. In addition, evidence for the synthesis ofproteins corresponding to these secondary ORFs of APV and RSV has notbeen reported.

The Fusion Protein (F) Gene

The F ORF of MPV is located adjacent to the M ORF, which ischaracteristic for members of the Metapneumovirus genus. The F gene ofMPV encodes a 639 aa protein, which is two aa residues longer than F ofAPV-C (Table 5). Analysis of the aa sequence revealed 81% homology withAPV-C, 67% with APV-A and B, 33-39% with Pneumovirus F proteins and only10-18% with other paramyxoviruses (Table 6). One of the conservedfeatures among F proteins of paramyxoviruses, and also seen in MPV isthe distribution of cysteine residues (Morrison, 1988; Yu et al., 1991).The Metapneumoviruses share 12 cysteine residues in F1 (7 are conservedamong all paramyxoviruses), and two in F2 (1 is conserved among allparamyxoviruses). Of the 3 potential N-linked glycosylation sitespresent in the F ORF of MPV, none are shared with RSV and two (position74 and 389) are shared with APV. The third, unique, potential N-linkedglycosylation site for MPV is located at position 206 (FIG. 11).

Despite the low sequence homology with other paramyxoviruses, the Fprotein of MPV revealed typical fusion protein characteristicsconsistent with those described for the F proteins of otherParamyxoviridae family members (Morrison, 1988). F proteins ofParamyxoviridae members are synthesized as inactive precursors (F0) thatare cleaved by host cell proteases which generate amino terminal F2subunits and large carboxy terminal F1 subunits. The proposed cleavagesite (Collins et al., 1996) is conserved among all members of theParamyxoviridae family. The cleavage site of MPV contains the residuesRQSR. Both arginine (E) residues are shared with APV and RSV, but theglutamine (Q) and serine (S) residues are shared with otherparamyxoviruses such as human parainfluenza virus type 1, Sendai virusand morbilliviruses (data not shown).

The hydrophobic region at the amino terminus of F1 is thought tofunction as the membrane fusion domain and shows high sequencesimilarity among paramyxoviruses and morbilliviruses and to a lesserextent the Pneumoviruses (Morrison, 1988). These 26 residues (position137-163, FIG. 11) are conserved between MPV and APV-C, which is inagreement with this region being highly conserved among theMetapneumoviruses (Naylor et al., 1998; Seal et al., 2000). As is seenfor the F2 subunits of APV and other paramyxoviruses, MPV revealed adeletion of 22 aa residues compared with RSV (position 107-128, FIG.11). Furthermore, for RSV and APV, the signal peptide and anchor domainwere found to be conserved within subtypes and displayed highvariability between subtypes (Plows et al., 1995; Naylor et al., 1998).The signal peptide of MPV (aa 10-35, FIG. 11) at the amino terminus ofF2 exhibits some sequence similarity with APV-C (18 out of 26 aaresidues are similar) and less conservation with other APVs or RSV. Muchmore variability is seen in the membrane anchor domain at the carboxyterminus of F1, although some homology is still seen with APV-C.

The 22K (M2) Protein

The M2 gene is unique to the Pneumovirinae and two overlapping ORFs havebeen observed in all Pneumoviruses. The first major ORF represents theM2-1 protein which enhances the processivity of the viral polymerase(Collins et al., 1995; Collins, 1996) and its readthrough of intergenicregions (Hardy et al., 1998; Fearns et al., 1999). The M2-1 gene forMPV, located adjacent to the F gene, encodes a 187 aa protein (Table 5),and reveals the highest (84%) homology with M2-1 of APV-C (Table 6).Comparison of all Pneumovirus M2-1 proteins revealed the highestconservation in the amino-terminal half of the protein (Collins et al.,1990; Zamora et al., 1992; Ahmadian et al., 1999), which is in agreementwith the observation that MPV displays 100% similarity with APV-C in thefirst 80 aa residues of the protein (FIG. 12A). The MPV M2-1 proteincontains 3 cysteine residues located within the first 30 aa residuesthat are conserved among all Pneumoviruses. Such a concentration ofcysteines is frequently found in zinc-binding proteins (Ahmadian et al.,1991; Cuesta et al., 2000).

The secondary ORFs (M2-2) that overlap with the M2-1 ORFs ofPneumoviruses are conserved in location but not in sequence and arethought to be involved in the control of the switch between virus RNAreplication and transcription (Collins et al., 1985; Elango et al.,1985; Baybutt et al., 1987; Collins et al., 1990; Ling et al., 1992;Zamora et al., 1992; Alansari et al., 1994; Ahmadian et al., 1999;Bermingham et al., 1999). For MPV, the M2-2 ORF starts at nucleotide 512in the M2-1 ORF (FIG. 7), which is exactly the same start position asfor APV-C. The length of the M2-2 ORFs are the same for APV-C and MPV,71 aa residues (Table 5). Sequence comparison of the M2-2 ORF (FIG. 12B)revealed 64% aa sequence homology between MPV and APV-C and only 44-48%aa sequence homology between MPV and APV-A and B (Table 6).

The Small Hydrophobic Protein (SH) ORF

The gene located adjacent to M2 of hMPV probably encodes a 183 aa SHprotein (FIGS. 1 and 7). There is no discernible sequence identitybetween this ORF and other RNA virus genes or gene products. This is notsurprising since sequence similarity between Pneumovirus SH proteins isgenerally low. The putative SH ORF of hMPV is the longest SH ORF knownto date (Table 1). The aa composition of the SH ORF is relativelysimilar to that of APV, RSV and PVM, with a high percentage of threonineand serine residues (22%, 18%, 19%, 20.0%, 21% and 28% for hMPV, APV,RSVA, RSV B, bRSV and PVM respectively). The SH ORF of hMPV contains 10cysteine residues, whereas APV SH contains 16 cysteine residues. The 511ORF of hMPV contains two potential N-linked glycosylation sites (aa 76and 121), whereas APV has one, RSV has two or three and PVM has four.

The hydrophilicity profiles for the putative hMPV SH protein and SH ofAPV and RSV revealed similar characteristics (FIG. 7B). The SH ORFs ofAPV and hMPV have a hydrophilic N-terminus, a central hydrophobic domainwhich can serve as a potential membrane spanning domain (aa 30-53 forhMPV), a second hydrophobic domain (aa 155-170) and a hydrophilicC-terminus. In contrast, RSV SH appears to lack the C-terminal part ofthe APV and hMPV ORFs. In all Pneumovirus SH proteins the hydrophobicdomain is flanked by basic aa residues, which are also found in the SHORF for hMPV (aa 29 and 54).

The Attachment Glycoprotein (G) ORF

The putative G ORF of hMPV is located adjacent to the putative SH geneand encodes a 236 aa protein (nt 6262-6972, FIG. 1). A secondary smallORF is found immediately following this ORF, potentially coding for 68aa residues (nt 6973-7179) but lacking a start codon. A third potentialORF in the second reading frame of 194 aa residues is overlapping withboth of these ORFs but also lacks a start codon (nt 6416-7000). This ORFis followed by a potential fourth ORF of 65 aa residues in the samereading frame (nt 7001-7198), again lacking a start codon. Finally, apotential ORF of 97 aa residues (but lacking a start codon) is found inthe third reading frame (nt 6444-6737, FIG. 1). Unlike the first ORF,the other ORFs do not have apparent gene start or gene end sequences(see below). Although the 236 aa G ORF probably represents at least apart of the hMPV attachment protein it cannot be excluded that theadditional coding sequences ale expressed as separate proteins or aspart of the attachment protein through some RNA editing event. It shouldbe noted that for APV and RSV no secondary ORFs after the primary G ORFhave been identified but that both APV and RSV have secondary ORFswithin the major ORF of G. However, evidence for expression of theseORFs is lacking and there is no sequence identity between the predictedaa sequences for different viruses (Ling et al., 1992). The secondaryORFs in hMPV G do not reveal characteristics of other G proteins andwhether the additional ORFs are expressed requires furtherinvestigation.

BLAST analyses with all ORFs revealed no discernible sequence identityat the nucleotide or aa sequence level with other known virus genes orgene products. This is in agreement with the low percentage sequenceidentity found for other G proteins such as those of hRSV A and B (53%)(Johnson et al., 1987) and APV A and B (38%) (Juhasz and Easton, 1994).

Whereas most of the hMPV ORFs resemble those of APV both in length andsequence, the putative G ORF of 236 aa residues of iMPV is considerablysmaller than the G ORF of APV (Table 1). The aa sequence revealed aserine and threonine content of 34%, which is even higher than the 32%for BSV and 24% for APV. The putative G ORF also contains 8.5% prolineresidues, which is higher than the 8% for RSV and 7% for APV. Theunusual abundance of proline residues in the G proteins of APV, RSV andiMPV has also been observed in glycoproteins of mucinous origin where itis a major determinant of the proteins three dimensional structure(Collins and Wertz, 1983; Wertz et al., 1985; Jentoft, 1990). The G ORFof hMPV contains five potential N-linked glycosylation sites, whereashRSV has seven, bRSV has five and APV has three to five.

The predicted hydrophilicity profile of hMPV G revealed characteristicssimilar to the other Pneumoviruses. The N-terminus contains ahydrophilic region followed by a short hydrophobic area (aa 33-53 forhMPV) and a mainly hydrophilic C-terminus (FIG. 8B). This overallorganization is consistent with that of an anchored type IItransmembrane protein and corresponds well with these regions in the Gprotein of APV and RSV. The putative G ORF of hMPV contains only 1cysteine residue in contrast to RSV and APV (5 and 20 respectively). Ofnote, only two of the four secondary ORFs in the G gene contained oneadditional cysteine residue and these four potential ORFs revealed12-20% serine and threonine residues and 6-11% proline residues.

The Polymerase Gene (L)

In analogy to other negative strand viruses, the last ORF of the MPVgenome is the RNA-dependent RNA polymerase component of the replicationand transcription complexes. The L gene of MPV encodes a 2005 aaprotein, which is 1 residue longer than the APV-A protein (Table 5). TheL protein of MPV shares 64% homology with APV-A, 42-44% with RSV, andapproximately 13% with other paramyxoviruses (Table 6). Poch et al.(1989; 1990) identified six conserved domains within the L proteins ofnon-segmented negative strand RNA viruses, from which domain IIIcontained the four core polymerase motifs that are thought to beessential for polymerase function. These motifs (A, B, C and D) are wellconserved in the MPV L protein: in motifs A, B and C: MPV shares 100%similarity with all Pneumoviruses and in motif D MPV shares 100%similarity-with APV and 92% with RSVs. For the entire domain I (aa627-903 in the L ORF), MPV shares 77% identity with APV, 61-62% with RSVand 23-27% with other paramyxoviruses (FIG. 15). In addition to thepolymerase motifs the Pneumovirus L proteins contain a sequence whichconforms to a consensus ATP binding motif K(X)₂₁GEGAGNM₂₀K (Stec, 1991).The MWV L ORF contains a similar motif as APV, in which the spacing ofthe intermediate residues is off by one: K(x)₂₂GEGAGN(X)₁₉K.

Phylogenetic Analyses

As an indicator for the relationship between MPV and members of thePneumovirinae, phylogenetic trees based on the N, P, M and F ORFs havebeen constructed previously (van den Hoogen et al., 2001) and revealed aclose relationship between MPV and APV-C. Because of the low homology ofthe MPV SH and G genes with those of other paramyxoviruses, reliablephylogenetic trees for these genes cannot be constructed. In addition,the distinct genomic organization between members of the Pneumovirus andMetapneumovirus genera make it impossible to generate phylogenetic treesbased on the entire genomic sequence. We therefore only constructedphylogenetic trees for the M2 and L genes in addition to thosepreviously published. Both these trees confirmed the close relationbetween APV and MPV within the Pneumovirinae subfamily (FIG. 16).

MPV Non-Coding Sequences

The gene junctions of the genomes of paramyxoviruses contain short andhighly conserved nucleotide sequences at the beginning and end of eachgene (gene start and gene end signals), possibly playing a role ininitiation and termination of transcription (Curran et al., 1999).Comparing the intergenic sequences between all genes of MPV revealed aconsensus sequence for the gene start signal of the N, P, M, F, M2 andG: GGGACAAGU (FIG. 17A), which is identical to the consensus gene startsignal of the Metapneumoviruses (Ling et al., 1992; Yu et al., 1992; Liet al., 1996; Bayon-Auboyer et al., 2000). The gene start signals forthe SH and L genes of MPV were found to be slightly different from thisconsensus (SH: GGGAUAAAU, (SEQ ID NO:167) L: GAGACAAAU). (SEQ ID NO:168)For APV the gene start signal of L was also found to be different fromthe consensus: AGGACCAAT (SEQ ID NO:169) (APV-A) (Randhawa et al., 1996)and GGGACCAGT (SEQ ID NO:170) (APV-D) (Bayon-Auboyer et al., 2000).

In contrast to the similar gene start sequences of MPV and APV, theconsensus gene end sequence of APV, UAGUUAAUU (SEQ ID NO:171) (Randhawaet al., 1996), could not be found in the MPV intergenic sequences. Therepeated sequence found in most genes, except the G-L intergenic region,was U AAAAA U/A/C (SEQ ID NO:172), which could possibly act as gene endsignal. However, since we sequenced viral RNA rather than mRNA,definitive gene end signals could not be assigned and thus requiresfurther investigation. The intergenic regions of Pneumoviruses vary insize and sequence (Curran et al., 1999; Blumberg et al., 1991; Collinset al., 1983). The intergenic regions of MPV did not reveal homologywith those of APV and RSV and range in size from 10 to 228 nucleotides(FIG. 17B). The intergenic region between the M and F ORFs of MPVcontains part of a secondary ORF, which starts in the primary M ORF (seeabove).

The intergenic region between SH and G contains 192 nucleotides, anddoes not appear to have coding potential based on the presence ofnumerous stop-codons in all three reading frames. The intergenic regionbetween G and L contains 241 nucleotides, which may include additionalORFs (see above). Interestingly, the start of the L ORF is located inthese secondary ORFs. Whereas the L gene of APV does not start in thepreceding G ORF, the L ORF of RSV also starts in the preceding M2 gene.At the 3′ and 5′ extremities of the genome of paramyxoviruses shortextragenic region are referred to as the leader and trailer sequences,and approximately the first 12 nucleotides of the leader and last 12nucleotides of the trailer are complementary, probably because they eachcontain basic elements of the viral promoter (Curran et al., 1999;Blumberg et al., 1991; Mink et al., 1986). The 3′ leader of MPV and APVare both 41 nucleotides in length, and some homology is seen in theregion between nucleotide 16 and 41 of both viruses (18 out of 26nucleotides) (FIG. 17B). As mentioned before the first 15 nucleotides ofthe MPV genomic map are based on a primer sequence based on the APVgenome. The length of the 5′ trailer of MPV (188 nucleotides) resemblesthe size of the RSV 5′ trailer (155 nucleotides), which is considerablylonger than that of APV (40 nucleotides). Alignments of the extreme 40nucleotides of the trailer of MPV and the trailer of APV revealed 21 outof 32 nucleotides homology, apart from the extreme 12 nucleotides whichrepresent primer sequences based on the genomic sequence of APV. Oursequence analyses revealed the absence of NS1 and NS2 genes at the 3′end of the genome and a genomic organization resembling the organizationof Metapneumoviruses (3′-N-P-M-F-M2-SH-G-L-5′). The high sequencehomology found between MPV and APV genes further emphasizes the closerelationship between these two viruses. For the N, P, M, F, M2-1 andM2-2 genes of MPV an overall amino acid homology of 79% is found withAPV-C. In fact, for these genes APV-C and MPV revealed sequencehomologies which are in the same range as sequence homologies foundbetween subgroups of other genera, such as RSV-A and B or APV-A and B.This close relationship between APV-C and MPV is also seen in thephylogenetic analyses which revealed MPV and APV-C always in the samebranch, separate from the branch containing APV-A and B. The identicalgenomic organization, the sequence homologies and phylogenic analysesare all in favor of the classification of MPV as the first member in theMetapneumovirus genus that is isolatable from mammals. It should benoted that the found sequence variation between different virus isolatesof MPV in the N, M, F and L genes revealed the possible existence ofdifferent genotypes (van den Hoogen et al., 2001). The closerelationship between MPV and APV-C is not reflected in the host range,since APV infects birds in contrast to MPV (van den Hoogen et al.,2001). This difference in host range may be determined by thedifferences between the SH and G proteins of both viruses that arehighly divergent. The SH and G proteins of MPV did not revealsignificant aa sequence homology with SH and G proteins of any othervirus. Although the amino acid content and hydrophobicity plots are infavor of defining these ORFs as SH and G, experimental data are requiredto assess their function. Such analyses will also shed light on the roleof the additional overlapping ORFs in these SH and G genes. In addition,sequence analyses on the SH and G genes of APV-C might provide moreinsight in the function of the SH and G proteins of MPV and theirrelationship with those of APV-C. The noncoding regions of MPV werefound to be fairly similar to those of APV. The 3′ leader and 5′ trailersequences of APV and MPV displayed a high degree of homology. Althoughthe lengths of the intergenic regions were not always the same for APVand MPV, the consensus gene start signals of most of the ORFs were foundto be identical. In contrast, the gene end signals of APV were not foundin the MPV genome. Although we did find a repetitive sequence (U AAAAAU/A/C) (SEQ ID NO:172) in most intergenic regions, sequence analysis ofviral mRNAs is required to formally delineate those gene end sequences.It should be noted that sequence information for 15 nucleotides at theextreme 3′ end and 12 nucleotides at the extreme 5′ end is obtained byusing modified rapid amplification of cDNA ends (RACE) procedures. Thistechnique has been proven to be successful by others for related viruses(IS. Randhawa, et al., Rescue of synthetic minireplicons establishes theabsence of the NS1 and NS2 genes from avian Pneumovirus, J. Virol. 71,9849-9854 (1997); M. A. Mink, et al., Nucleotide sequences of the 3′leader and 5′ trailer regions of human respiratory syncytial virusgenomic RNA, Virology 185, 615-24 (1991).) To determine the sequence ofthe 3′ vRNA leader sequence, a homopolymer A tail is added to purifiedvRNA using poly-A-polymerase and the leader sequence subsequentlyamplified by PCR using a poly-T primer and a primer in the N gene. Todetermine the sequence of the 5′ vRNA trailer sequence, a cDNA copy ofthe trailer sequence is made using reverse transcriptase and a primer inthe L gene, followed by homopolymer dG tailing of the CDNA with terminaltransferase. Subsequently, the trailer region is amplified using apoly-C primer and a primer in the L gene. As an alternative strategy,vRNA is ligated to itself or synthetic linkers, after which the leaderand trailer regions are amplified using primers in the L and N genes andlinker-specific primers. For the 5′ trailer sequence directdideoxynucleotide sequencing of purified vRNA is also feasible(Randhawa, 1997). Using these approaches, we can analyze the exactsequence of the ends of the hMPV genome. The sequence informationprovided here is of importance for the generation of diagnostic tests,vaccines and antivirals for MPV and MPV infections.

Materials and Methods Sequence Analysis

Virus isolate 00-1 was propagated to high titers (approximately 10,000TCID50/ml) on tertiary monkey kidney cells as described previously (vanden Hoogen et al., 2001). Viral RNA was isolated from supernatants frominfected cells using a High Pure RNA Isolating Kit according toinstructions from the manufacturer (Roch Diagnostics, Almere, TheNetherlands). Primers were designed based on sequences publishedpreviously (van den Hoogen et al., 2001) in addition to sequencespublished for the leader and trailer of APV/RSV (Randhawa et al., 1997;Mink et al., 1991) and are available upon request. RT-PCR assays wereconducted with viral RNA, using a one-tube assay in a total volume of 50μl with 50 mM iris pH 8.5, 50 mM NaCl, 4.5 mM MgCl₂, 2 mM DTT, 1 μMforward primer, 1 μM reverse primer, 0.6 mM dNTPs, 20 units RNAsin(Promega, Leiden, The Netherlands), 10 U AMV reverse transcriptase(Promega, Leiden, The Netherlands), and 5 units Taq Polymerase (PEApplied Biosystems, Nieuwerkerk aan de IJssel, The Netherlands). Reversetranscription was conducted at 42° C. for 30 minutes, followed by 8minutes inactivation at 95° C. The cDNA was amplified during 40 cyclesof 95° C., 1 min.; 42° C., 2 min. 72° C., 3 min. with a final extensionat 72° C. for 10 minutes. After examination on a 1% agarose gel, theRT-PCR products were purified from the gel using a Qiaquick GelExtraction kit (Qiagen, Leusden, The Netherlands) and sequenced directlyusing a Dyenamic ET terminator sequencing kit (Amersham PharmaciaBiotech, Roosendaal, the Netherlands) and an ABI 373 automatic DNAsequencer (PE Applied Biosystem, Nieuwerkerk aan den IJssel, theNetherlands), according to the instructions of the manufacturer.

Sequence alignments were made using the clustal software packageavailable in the software package of BioEdit version 5.0.6. (on theInternet at jwbrown.mbio.ncsu.edu/Bioedit//bioedit.html; Hall, 1999).

Phylogenetic Analysis

To construct phylogenetic trees, DNA sequences were aligned using theClustalW software package and maximum likelihood trees were generatedusing the DNA-ML software package of the Phylip 3.5 program using 100bootstraps and 3 jumbles. Bootstrap values were computed for consensustrees created with the consense package (Felsenstein, 1989).

The MPV genomic sequence is available from Genbank under accessionnumber AF371337. All other sequences used here are available fromGenbank under accession numbers AB046218 (measles virus, all ORFs),NC-001796 (human parainfluenza virus type 3, all ORFs), NC-001552(Sendai virus, all ORFs), X57559 (human parainfluenza virus type 2, allORFs), NC-002617 (New Castle Disease virus, all ORFs), NC-002728 (Nipahvirus, all ORFs), NC-001989 (bRSV, all ORFs), M11486 RSV A, all ORFsexcept L), NC-001803 (HRSV, L ORM, NC-001781 (hRSV B, all ORFs), D10331(PVM, N ORF), U09649 (PVM, P ORF), U66893 (PVM, M ORF), U66893 (PVM, SHORF), D11130 (PVM, G ORF), D11128 (F ORF). The PVM M2 ORF was taken fromAhmadian (1999), AF176590 (APV-C, N ORF), U39295 (APV-A, N ORF), U39296(APV-B, N ORF), AF262571 (APV-C, M ORM), U37586 (APV-B, M ORF), X58639(APV-A, M ORF), AF176591 (APV-C, P ORF), AF325443 (APV-B, P ORF), U22110(APV-A, P ORF), AF187152 (APV-C, F ORF), Y14292 (APV-B, F ORF), D00850(APV-A, F ORF), AF176592 (APV-C, M2 ORF), AF35650 (APV-B, M2 ORF),X63408 (APV-A, M2 ORF), U65312 (APV-A, L ORF), S40185 (APV-A, SH ORF).

TABLE 5 Lengths of the ORFs of MPV and other paramyxoviruses N¹ P M FM2-1 M2-2 SH G L MPV 394 294 254 539 187 71 183 236 2005 APV A 391 278254 538 186 73 174 391 2004 APV B 391 279 254 538 186 73 —² 414 —² APV C394 294 254 537 184 71 —² —² —² APV D —² —² —² —² —² —² —² 389 —² hRSV A391 241 256 574 194 90 64 298 2165 hRSV B 391 241 249 574 195 93 65 2992166 bRSV 391 241 256 569 186 93 81 257 2162 PVM 393 295 257 537 176 7792 396 —² others³ 418-542 225-709 335-393 539-565 —⁴ —⁴ —⁴ —⁴ 2183-2262Footnotes: ¹length in amino acid residues. ²sequences not available³others: human parainfluenza virus type 2 and 3, Sendai virus, measlesvirus, nipah virus, phocine distemper virus, and New Castle Diseasevirus. ⁴ORF not present in viral genome

TABLE 6 Amino acid sequence identity between the ORFs of MPV and thoseof other paramyxoviruses¹ N P M F M2-1 M2-2 L APV A 69 55 78 67 72 26 64APV B 69 51 76 67 71 27 —² APV C 88 68 87 81 84 56 —² hRSV A 42 24 38 3436 18 42 hRSV B 41 23 37 33 35 19 44 bRSV 42 22 38 34 35 13 44 PVM 45 2637 39 33 12 —² others³ 7-11 4-9 7-10 10-18 —⁴ —⁴ 13-14 Footnotes: ¹Nosequence homologies were found with known G and SH proteins and werethus excluded ²Sequences not available. ³See list in table 5, footnote3. ⁴ORF absent in viral genome.

REFERENCES

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Further references used with Example 4.

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Boursnell, and D.    Cavanagh (1991). Deduced amino acid sequence of the fusion    glycoprotein of turkey rhinotracheitis virus has greater identity    with that of human respiratory syncytial virus, a Pneumovirus, than    that of paramyxoviruses and morbilliviruses. J. Gen. Virol.    72:75-81.-   Yu Q., P. J. Davis, J. Li, and D. Cavanagh (1992). Cloning and    sequencing of the matrix protein (M) gene of turkey rhinotracheitis    virus reveal a gene order different from that of respiratory    syncytial virus. Virology 186:426-34.-   Zamora M. and S. K. Samal (1992). Sequence analysis of M2 mRNA of    bovine respiratory syncytial virus obtained from an F-M2 dicistronic    mRNA suggests structural homology with that of human respiratory    syncytial virus. J. Gen. Virol. 73:737-41.-   Primers used for RT-PCR detection of known paramyxo-viruses. Primers    for hPIV-1 to 4, mumps, measles, Tupaia, Mapuera and Hendra are    developed in house and based on alignments of available sequences.    Primers for New Castle Disease Virus are taken from Seal, J., J. et    al; Clin. Microb., 2624-2630, 1995. Primers for Nipah and general    paramyxovirus-PCR are taken from: Chua, K. B., et al; Science, 288    26 May 2000.

located in Virus primers protein HPIV-1 fwd5′-TGTTGTCGAGACTATTCCAA-3′(SEQ ID NO: 132) HN Rev5′-TGTTG(T/A)ACCAGTTGCAGTCT-3′ (SEQ ID NO: 133) HPIV-2 Fwd5′-TGCTGCTTCTATTGAGAAACGCC-3′ (SEQ ID NO: 134) N Rev5′-GGTGAC/T TC(T/C)AATAGGGCCA-3′ (SEQ ID NO: 135) HPIV-3 Fwd5′-CTCGAGGTTGTCAGGATATAG-3′ (SEQ ID NO: 136) HN Rev5′-CTTTGGGAGTTGAACACAGTT-3′ (SEQ ID NO: 137) HPIV-4 Fwd5′-TTC(A/G)GTTTTAGCTGCTTACG-3′ (SEQ ID NO: 138) N Rev5′-AGGCAAATCTCTGGATAATGC-3′ (SEQ ID NO: 139) Mumps Fwd5′-TCGTAACGTCTCGTGACC-3′ (SEQ ID NO: 140) SH Rev5′-GGAGATCTTTCTAGAGTGAG-3′ (SEQ ID NO: 141) NDV Fwd5′-CCTTGGTGAiTCTATCCGIAG-3′ (SEQ ID NO: 142) F Rev5′-CTGCCACTGCTAGTTGiGATAATCC-3′ (SEQ ID NO: 143) Tupaia Fwd5′-GGGCTTCTAAGCGACCCAGATCTTG-3′ (SEQ ID NO: 144) N Rev5′-GAATTTCCTTATGGACAAGCTCTGTGC-3′ (SEQ ID NO: 145) Mapuera Fwd5′-GGAGCAGGAACTCCAAGACCTGGAG-3′ (SEQ ID NO: 146) N Rev5′-GCTCAACCTCATCACATACTAACCC-3′ (SEQ ID NO: 147) Hendra Fwd5′-GAGATGGGCGGGCAAGTGCGGCAACAG-3′ (SEQ ID NO: 148) N Rev5′-GCCTTTGCAATCAGGATCCAAATTTGGG-3′ (SEQ ID NO: 149) Nipah Fwd5′-CTGCTGCAGTTCAGGAAACATCAG-3′ (SEQ ID NO: 150) N Rev5′-ACCGGATGTGCTCACAGAACTG-3′ (SEQ ID NO: 151) HRSV Fwd5′-TTTGTTATAGGCATATCATTG-3′ (SEQ ID NO: 152) F Rev5′-TTAACCAGCAAAGTGTTA-3′ (SEQ ID NO: 153) Measles Fwd5′-TTAGGGCAAGAGATGGTAAGG-3′(SEQ ID NO: 154) N Rev5′-TTATAACAATGATGGAGGG-3′ (SEQ ID NO: 155) General Paramyxoviridae: Fwd5′-CATTAAAAAGGGCACAGACGC-3′ (SEQ ID NO: 156) P Rev5′-TGGACATTCTCCGCAGT-3′ (SEQ ID NO: 157) Primers for RAP-PCR: ZF1:5′-CCCACCACCAGAGAGAAA-3′ (SEQ ID NO: 158) ZF4: 5′-ACCACCAGAGAGAAACCC-3′(SEQ ID NO: 159) ZF7: 5′-ACCAGAGAGAAACCCACC-3′ (SEQ ID NO: 160) ZF10:5′-AGAGAGAAACCCACCACC-3′ (SEQ ID NO: 161) ZE13: 5′-GAGAAACCCACCACCAGA-3′(SEQ ID NO: 162) ZF16: 5′-AAACCCACCACCAGAGAG-3′ (SEQ ID NO: 163) CS1:5′-GGAGGCAAGCGAACGCAA-3′ (SEQ ID NO: 164) CS4: 5′-GGCAAGCGAACGCAAGGA-3′(SEQ ID NO: 165) CS7: 5′-AAGCGAACGCAAGGAGGC-3′ (SEQ ID NO: 101) CS10:5′-CGAACGCAAGGAGGCAAG-3′ (SEQ ID NO: 102) CS13: 5′-ACGCAAGGAGGCAAGCGA-3′(SEQ ID NO: 103) CS16: 5′-CAAGGAGGCAAGCGAACG-3′ (SEQ ID NO: 104)

20 fragments successfully purified and sequenced:

10 fragments found with sequence homology in APV

Fragment 1 ZF 7, 335 bp N gene Fragment 2 ZF 10, 235 bp N gene Fragment3 ZF 10, 800 bp M gene Fragment 4 CS 1, 1250 bp F gene Fragment 5 CS 10,400 bp F gene Fragment 6 CS 13, 1450 bp F gene Fragment 7 CS 13, 750 bpF gene Fragment 8 ZF 4, 780 bp L gene (protein level) Fragment 9 ZF 10,330 bp L gene (protein level) Fragment 10 ZF 10, 250 bp L gene (proteinlevel)

Primers used for RAP-PCR amplification of nucleic acids from theprototype isolate.

Example 5 Further Exploration of the Two Subtypes of hMPV

Based on phylogenetic analysis of the different isolates of hMPVobtained so far, two genotypes have been identified with virus isolate00-1 being the prototype of genotype A and isolate 99-1 the prototype ofgenotype B.

We hypothesize that the genotypes are related to subtypes and thatre-infection with viruses from both subgroups occur in the presence ofpre-existing immunity and the antigenic variation may not be strictlyrequired to allow re-infection.

Furthermore, hMPV appears to be closely related to avian Pneumovirus, avirus primarily found in poultry. The nucleotide sequences of bothviruses show high percentages of homology, with the exception of the SHand G proteins. Here we show that the viruses are cross-reacting intests, which are based primarily on the nucleoprotein and matrixprotein, but they respond differently in tests, which are based on theattachment proteins. The differences in virus neutralization titersprovide further proof that the two genotypes of hMPV are two differentserotypes of one virus, where APV is a different virus.

The Cross-Reaction Between the Two Serotypes and the Cross-ReactionBetween APV and hMPV Methods

Protocol for IgG, IgA and IgM antibody detection for hMPV:

The indirect IgG EIA for hMPV was performed in microtiter platesessentially as described previously (Rothbarth, P. H. et al., 1999;Influenza virus serology-a comparative study. J. of Vir. Methods 78(1999) 163-169.

Briefly, concentrated hMPV was solubilized by treatment with 1% TRITON®X-100 an coated for 16 hours at room temperature into microtiter platesin PBS after determination of the optimal working dilution bycheckerboard titration. Subsequently, 100 μl volumes of 1:100 dilutedhuman serum samples in EIA buffer were added to the wells and incubatedfor 1 hour at 37° C. Binding of human IgG was detected by adding a goatanti-human IgG peroxidase conjugate (Biosource, USA). Adding TMB assubstrate developed plates and OD was measured at 450 nm. the resultswere expressed as the S(ignal)/N(egative) ratio of the OD. A serum wasconsidered positive for IgG, if the SIN ratio was beyond the negativecontrol plus three times the standard.

hMPV antibodies of the IgM and IgA classes were detected in sera bycapture EIA essentially as described previously (P. H. Rothbarth et al.1999, Influenza virus serology-a comparative study. J. Vir. Methods 78(1999) 163-169). For the detection of IgA and IgM commercially availablemicrotiter plates coated with anti-human IgM or IgA specific monoclonalantibodies were used. Sera were diluted 1:100 and after incubation of 1hour at 37° C., an optimal working dilution of hMPV is added at eachwell (100 μl). Incubated 1 hour 37° C. After washing polyclonal antihMPV labeled with peroxidase was added, the plate was incubated 1 hour37° C. Adding TMB as substrate developed plates and OD was measured at450 nm. the results were expressed as the S(ignal)/N(egative) ratio ofthe OD. A serum was considered positive for IgG, if the S/N ratio wasbeyond the negative control plus three times the standard.

AVP antibodies were detected in an AVP inhibition assay. Protocol forAPV inhibition test is included the APV-Ab SVANOVIR® enzyme immunoassaywhich is manufactured by SVANOVA Biotech AB, Uppsal Science Park GluntenSE-751 83 Uppsala Sweden. The results were expressed as theS(ignal)/N(egative) ratio of the OD. A serum was considered positive forIgG, if the S/N ratio was beyond the negative control plus three timesthe standard.

1. Guinea Pigs

A. (Re)Infection of Guinea Digs with Both Subtypes of hMPV

Virus isolates ned/00/01 (subtype A) and ned/99/01 (subtype B) have beenused to inoculate 6 guinea pigs per subtype (intratracheal, nose andeyes).

-   -   6 GPs infected with hMPV 00-1 (10e6, 6 TCID50)    -   6 GPs infected with hMPV 99-1 (10e4, 1 TCED50)    -   54 Days after the primary infection, the guinea pigs have been        inoculated with the homologous and heterologous subtypes (10e4        TCED50/ml):    -   2 guinea pigs: 1^(st) infection 00-1; 2^(nd) 99-1 (heterologous)    -   3 guinea pigs: 1^(st) infection 00-1; 2^(nd) 00-1 (homologous)    -   2 guinea pigs: 1^(st) infection 99-1; 2^(nd) 00-1 (heterologous)    -   3 guinea pigs: 1^(st) infection 99-1; 2^(nd) 99-1 (homologous)

Throat and nose swabs have been collected for 12 days (1^(st) infection)or 8 days (2^(nd) infection) post infection, and have been tested forpresence of the virus by RT-PCR assays.

Results of RT-PCR Assay: FIG. 29

Summary of results: guinea pigs inoculated with virus isolate ned/00/01show infection of the upper respiratory tract day 1 to 10 postinfection. Guinea pigs inoculated with ned/99/01 show infection of theupper respiratory tract day 1 to 5 post infection. Infection withned/99/01 appears to be less severe than infection with ned/00/01. Asecond inoculation of the guinea pigs with the heterologous virusresults in re-infection in 3 out of 4 guinea pigs and with thehomologous virus in 2 out of 6 guinea pigs. No or only little clinicalsymptoms were noted in those animals that became re-infected, and noclinical symptoms were seen in those animals that were protected againstthe re-infections, demonstrating that even with wild-type virus, aprotective effect of the first infection is evident, showing thepossible use of heterologous (and of course homologues) isolates as avaccine, even in an unattenuated form.

Both subtypes of hMPV are able to infect guinea pigs, although infectionwith subtype B (ned/99/01) seems less severe (shorter period of presenceof the virus in nose and throat) than infection with subtype A(ned/00/01). This may be due to the higher dose given for subtype A, orto the lower virulence of subtype B.

Although the presence of pre-existing immunity does not completelyprotect against re-infection with both the homologous and heterologousvirus, the infection appears to be less prominent in that a shorterperiod of presence of virus was noted and not all animals became viruspositive.

B. Serology of Guinea Pigs Infected with Both Subtypes of hMPV

At day 0, 52, 70, 80, 90, 110, 126 and 160 sera were collected from theguinea pigs and tested at a 1:100 dilution in a whole virus ELISAagainst ned/00/01 and ned/99/01 antigen.

FIGS. 30A and 30B: IgG response against ned/00/01 and ned/99/01 for eachindividual guinea pig

FIG. 31: Specificity of the ned/00/01 and ned/99/01 ELISA. Only datafrom homologous reinfected guinea pigs have been used.

FIG. 32: Mean IgG response against ned/00/01 and ned/99/01 ELISA of 3homologous (00-1/00-1), 2 homologous (99-1/99-1), 2 heterologous(99-1/00-1) and 2 heterologous (00-1/99-1) infected guinea pigs.

Summary of Results:

Only a minor difference in response to the two different ELISAs isobserved. Whole virus ELISA against 00-1 or 99-1 cannot be used todiscriminate between the two subtypes.

C. Reactivity of Sera Raised Against hM:PV in Guinea Pigs with APVAntigen

Sera collected from the infected guinea pigs have been tested with anAPV inhibition ELISA.

FIG. 33: Mean percentage of APV inhibition of hMPV-infected guinea pigs.

Summary of Results:

Sera raised against hMPV in guinea pigs, react in the APV inhibitiontest in a same manner as they react in the hbNV IgG ELISAs.

Sera raised against ned/99/01 reveal a lower percentage of inhibition inthe APV inhibition ELISA than sera raised against ned/00/01. Guinea pigsinfected with ned/99/01 might have a lower titer (as is seen in the hMPVEUISAs) or the cross-reaction of ned/99/01 with APV is less than that ofned/00/01. Nevertheless, the APV-Ab inhibition ELISA can be used todetect hMPV antibodies in guinea pigs.

D. Virus Neutralization Assays with Sera Raised Against hMPV in GuineaPigs.

Sera collected at day 0, day 52, 70 and 80 post infection were used in avirus (cross) neutralization assay with ned/00/01, ned/99/01 and APV-C.Starting dilution was 1 to 10 and 100 TCID50 virus per well was used.After neutralization, the virus was brought on tMK cells, 15 minutescentrifuged at 3500 RPM, after which the media was refreshed.

The APV tests were grown for 4 days and the hMPV tests were grown for 7days. Cells were fixed with 80% aceton, and IFAs were conducted withmonkey-anti hMPV fitc labeled. Wells that were negative in the stainingwere considered as the neutralizing titer. For each virus a 10-logtitration of the virus stock and 2 fold titration of the workingsolution was included.

FIG. 34: Virus neutralization titers of ned/00/01 and ned/99/01 infectedguinea pigs against ned/00/01, ned/99/01 and APV-C

2. Cynomolgus Macaques

A. (Re)Infection of Cynomolgus Macaques with Both Subtypes of hMPV

Virus isolates ned/00/01 (subtype A) and ned/99/01 (subtype B) (1*5TCID50) have been used to inoculate 2 cynomolgus macaques per subtype(intratracheal, nose and eyes). Six months after the primary infection,the macaque have been inoculated for the second time with ned/00/01.Throat swabs have been collected for 14 days (1^(st) infection) or 8days (2^(nd) infection) post infection, and have been tested forpresence of the virus by RT-PCR assays.

FIG. 35: Results of RT-PCR assays on throat swabs of cynomolgus macaquesinoculated (twice) with ned/00/01.

Summary of Results:

Summary of results: cynomolgus macaques inoculated with virus isolatened/00/01 show infection of the upper respiratory tract day 1 to 10 postinfection. Clinical symptoms included a suppurative rhinitis. A secondinoculation of the macaques with the homologous virus results inre-infection, as demonstrated by PCR, however, no clinical symptoms wereseen.

B. Serology on Sera Collected of hMPV-Infected Cynomolgus Macaques.

From the macaques which received ned/00/01 sera were collected during 6months after the primary infection (re-infection occurred at day 240 formonkey 3 and day 239 for monkey 6).

Sera were used to test for the presence of IgG antibodies against eitherned/00/01 or APV, and for the presence against IgA and IgM antibodiesagainst ned/00/01.

Results: FIG. 36A: IgA, IgM and IgG response against ned/00/01 of 2cynomolgus macaques (re)infected with ned/00/01.

FIG. 36B: IgG response against APV of 2 cynomolgus macaques infectedwith ned/00/01.

Summary of Results:

Two macaques have been successfully infected with ned/00/01 and in thepresence of antibodies against ned/00/01 been reinfected with thehomologous virus. The response to IgA and IgM antibodies shows the raisein IgM antibodies after the first infection, and the absence of it afterthe reinfection. IgA antibodies are only detected after there-infection, showing the immediacy of the immune response after a firstinfection. Sera raised against hMPV in macaques which were tested in anAPV inhibition ELISA show a similar response as to the hMPV IgG EUSA

Discussion/Conclusion

hMPV antibodies in cynomolgus macaques are detected with the APVinhibition ELISA with a similar sensitivity as with an hMPV ELISA, andtherefore the APV inhibition EIA is suitable for testing human samplesfor the presence of hMPV antibodies.

C. Virus (Cross) Neutralization Assays with Sera Collected fromhMPV-Infected Cynomolgus Macaques

Summary of results: The sera taken from day 0 to day 229 post primaryinfection show only low virus neutralization titers against ned/00/01(0-80), the sera taken after the secondary infection show highneutralization titers against ned/00/01: >1280. Only sera taken afterthe secondary infection show neutralization titers against ned/99/01(80-640), and none of the sera neutralize the APV C virus.

There is no cross-reaction between APV-C and hMPV in virus (cross)neutralization assays, where there is a cross-reaction between ned/00/01and ned/99/01 after a boost of the antibody response.

3. Humans

Sera of patients ranging in age of <6 months to >20 years of age havepreviously been tested in IFA and virus neutralization assays againstned/00/01. (See Table 1 of patent.)

Here we have tested a number of these sera for the presence of IgG, IgMand IgA antibodies in an ELISA against ned/00/01, and we tested thesamples in the APV inhibition ELISA.

Results: FIG. 37 Comparison of the use of the hMPV ELISA and the APVinhibition ELISA for the detection of IgG antibodies in human sera,there is a strong correlation between the IgG hMPV test and the APV-Abtest, therefore the APV-Ab test is essentially able to detect IgGantibodies to hmPV in humans.

4. Poultry

96 chickens have been tested in both the APV inhibition ELISA and thened/00/01 ELISA for the presence of IgG antibodies against APV.

Summary of results: Both the hMPV ELISA and the APV inhibition ELISAdetect antibodies against APV (data not shown).

Summary of Results.

We found two genotypes of hMPV with ned/00/01 being the prototype ofsubgroup A and ned/99/01 the prototype of subgroup B.

“According to classical serological analyses (as, for example, known R.I. B. Francki, C. M. Fauquet, D. L. Knudson, and F. Brown,Classification and nomenclature of viruses, Fifth report of theinternational Committee on Taxonomy of Viruses, Arch. Virol., 1991.Supplement 2: p. 140-144), two subtypes can be defined on the basis ofits immunological distinctiveness, as determined by quantitativeneutralization assays with animal antisera. Two distinct serotypes haveeither no cross-reaction with each other or show a homologous-toheterologous titer ratio >16 in both directions. If neutralization showsa certain degree of cross-reaction between two viruses in either or bothdirections (homologous-to-heterologous titer ration of eight or 16),distinctiveness of serotype is assumed if substantialbiophysical/biochemical differences of DNAs exist. If neutralizationshows a distinct degree of cross-reaction between two viruses in eitheror both directions (homologous-to-heterologous titer ration of smallerthan eight), identity of serotype of the isolates under study isassumed.”

For RSV, it is known that re-infection occurs in the presence ofpre-existing immunity (both homologous and heterologous). Infection ofguinea pigs and cynomolgus macaques with both the homologous andheterologous serotypes of hMPV revealed that this is also true for hMPV.In addition, IgA and IgM ELISAs against hMPV revealed the reaction ofIgA antibodies only occurs after re-infection. Sera raised against hMPVor APV respond in an equal way in APV and hMPV ELISAs. From thenucleotide sequence comparisons, it is known that the viruses show about80% amino acid homology for the N, P, M, and F genes. In ELISAs, the Nand M proteins are the main antigens to react. Virus neutralizationassays (known to react against the surface glycoproteins G, SH and F)show a difference between the two different sera. Although APV en hMPVcross-react in ELISAs, phylogenetic analyses of the nucleotide sequencesof hMPV and APV, the differences in virus neutralization titers of seraraised against the two different viruses, and the differences in hostusage again reveal that APV-C and hMPV are two different viruses. Basedon the results we speculate that hMPV infection in mammals is possible aresult of a zoonotic event from birds to mammals. But the virus hasadapted in such a way (i.e., the G and SH proteins) that a return (frommammals to birds) zoonotic event seems unlikely, considering thepresence of AVP in birds.

Addendum Background Information on Pneumovirinae

The family of Paramyxoviridae contains two subfamilies: theParamyxovirinae and the Pneumovirinae. The subfamily Pneumovirinaeconsists of two genera: Pneumovirus and Metapneumovirus. The genusPneumovirus contains the human, bovine, ovine and caprine respiratorysyncytial viruses and the pneumonia virus of mice (PVM). The genusMetapneumovirus contains the avian Pneumoviruses (APV, also referred toas TRTV).

The classification of the genera in the subfamily Pneumovirinae is basedon classical virus characteristics, gene order and gene constellation.Viruses of the genus Pneumovirus are unique in the family ofParamyxoviridae in having two nonstructural proteins at the 3′ end ofthe genome (3′-NS1-NS2-N-P-M-SH-G-F-M2-L-5′). In contrast, viruses inthe genus Metapneumovirus lack the NS1 and NS2 genes and theorganization of genes between the M and L coding regions is different:3′-N-P-M-F-M2-SH-G-L-5′.

All members of the subfamily Paramyxovirinae have hemagglutinatingactivity, but this function is not a defining feature for the subfamilyPneumovirinae, being absent in RSV and APV but present in PMV.Neuraminidase activity is present in members of the genera Paramyxovirusand Rubulavirus (subfamily Paramyxovirinae) but is absent in the genusMorbillivirus (subfamily Paramyxovirinae) and the genera Pneumovirus andMetapneumovirus (subfamily Pneumovirinae).

A second distinguishing feature of the subfamily Pneumovirinae is theapparent limited utilization of alternative ORFs within mRNA by RSV. Incontrast, several members of the subfamily Paramyxovirinae, such asSendai and Measles viruses, access alternative ORFs within the mRNAencoding the phosphoprotein (P) to direct the synthesis of a novelprotein.

The G protein of the Pneumovirinae does not have sequence relatedness orstructural similarity to the HN or H proteins of Paramyxovirinae and isonly approximately half the size of their chain length. In addition, theN and P proteins are smaller than their counterparts in theParamyxovirinae and lack unambiguous sequence homology. Mostnonsegmented negative-stranded RNA viruses have a single matrix (M)protein.

Members of the subfamily Pneumovirinae are an exception in having twosuch proteins, M and M2. The M protein is smaller than itsParamyxovirinae counterparts and lacks sequence relatedness withParamyxovirinae.

When grown in cell cultures, members of the subfamily Pneumovirinae showtypical cytopathic effects; they induce characteristic syncytiaformation of cells. (Collins, 1996). The subfamily Pneumovirinae, genusPneumovirus

hRSV is the type-species of the genus Pneumovirus and is a major andwidespread cause of lower respiratory tract illness during infancy andearly childhood (Selwyn, 1990). In addition, hRSV is increasinglyrecognized as an important pathogen in other patient groups, includingimmune compromised individuals and the elderly. RSV is also an importantcause of community-acquired pneumonia among hospitalized adults of allages (Englund, 1991; Falsey, 2000; Dowell, 1996). Two major antigenictypes for RSV (A and B) have been identified based on differences intheir reactivity with monoclonal and polyclonal antibodies and bynucleic acid sequence analyses (Anderson, 1985; Johnson, 1987;Sullender, 2000). In particular the G protein is used in distinguishingthe two subtypes. RSV-A and B share only 53% amino acid sequencehomology in G, whereas the other proteins show higher homologies betweenthe subtypes (Table 1) (Collins, 1996).

Detection of RSV infections has been described using monoclonal andpolyclonal antibodies in immunofluorescence techniques (DIF, IFA), virusneutralization assays and ELISA or RT-PCR assays (Rothbarth, 1988; VanMilaan, 1994; Coggins, 1998). Closely related to hRSV are the bovine(bRSV), ovine (oRSV) and caprine RSV (oRSV), from which bRSV has beenstudied most extensively. Based on sequence homology with HRSV, theruminant RSVs are classified within the Pneumovirus genus, subfamilyPneumovirinae (Collins, 1996). Diagnosis of ruminant RSV infection andsubtyping is based on the combined use of serology, antigen detection,virus isolation and RT-PCR assays (Uttenthal, 1996; Valarcher, 1999;Oberst, 1993; Vilcek, 1994).

Several analyses on the molecular organization of bRSV have beenperformed using human and bovine antisera, monoclonal antibodies andcDNA probes. These analyses revealed that the protein composition ofhRSV and bRSV are very similar and the genomic organization of bRSVresembles that of hRSV. For both bRSV and HRSV, the G and F proteinsrepresent the major neutralization and protective antigens. The Gprotein is highly variable between the hRSV subtypes and between hRSVand bRSV (53 and 28% respectively) (Prozzi, 1997; Lerch, 1990). The Fproteins of hRSV and bRSV strains present comparable structuralcharacteristics and antigenic relatedness. The F protein of bRSV shows80-81% homology with hRSV, while the two hRSV subtypes share 90%homology in F (K. Walravens, 1990).

Studies based on the use of hRSV and bRSV specific monoclonal antibodieshave suggested the existence of different antigenic subtypes of bRSV.Subtypes A, B, and AB are distinguished based on reaction patterns ofmonoclonal antibodies specific for the G protein (Furze, 1994; Prozzi,1997; Elvander, 1998). The epidemiology of bRSV is very similar to thatof hRSV. Spontaneous infection in young cattle is frequently associatedwith severe respiratory signs, whereas experimental infection generallyresults in milder disease with slight pathologic changes (Elvander,1996).

RSV has also been isolated from naturally infected sheep (oRSV)(LeaMaster, 1983) and goats (cRSV) (Lehmkuhl, 1980). Both strains share96% nucleotide sequence with the bovine RSV and are antigenicallycross-reacting. Therefore, these viruses are also classified within thePneumovirus genus.

A distinct member of the subfamily Pneumovirinae, genus Pneumovirus isthe Pneumonia virus of mice (PVM).

PVM is a common pathogen in laboratory animal colonies, particularlythose containing atymic mice. The naturally acquired infection isthought to be asymptomatic, though passage of virus in mouse lungsresulted in overt signs of disease ranging from an upper respiratorytract infection to a fatal pneumonia (Richter, 1988; Weir, 1988).

Restricted serological cross-reactivity between the nucleocapsid protein(N) and the phosphoprotein (P) of PVM and hRSV has been described butnone of the external proteins show cross-reactivity, and the viruses canbe distinguished from each other in virus neutralization assays(Chambers, 1990a; Gimenez, 1984; Ling, 1989a). The glycoproteins of PVMappear to differ from those of other paramyxoviruses and resemble thoseof RSV in terms of their pattern of glycosylation. They differ, however,in terms of processing. Unlike RSV, but similar to the otherparamyxoviruses, PVM has hemagglutinating activity with murineerythrocytes, for which the G protein appears to be responsible since amonoclonal antibody to this protein inhibits hemagglutination (Ling,1989b).

The genome of PVM resembles that of H-RSV, including two nonstructuralproteins at, its 3′ end and a similar genomic organization (Chambers,1990a; Chambers, 1990b). The nucleotide sequences of the PVM NS1/NS2genes are not detectably homologous with those of hRSV (Chambers, 1991).Some proteins of PVM show strong homology with hRSV (N: 60%, and F: 38to 40%) while G is distinctly different (the amino acid sequence is 31%longer) (Barr, 1991; Barr, 1994; Chambers, 1992). The PVM P gene, butnot that of RSV or APV, has been reported to encode a second ORF,representing a unique PVM protein (Collins, 1996). New PVM isolates areidentified by virus isolation, hemagglutination assays, virusneutralization assay and various immuno-fluorescence techniques.

Table with addendum: Amino acid homology between the different viruseswithin the genus Pneumovirus of the subfamily Pneumovirinae oRSV vs.bRSV vs. bRSV vs. PVM vs. Gene hSRVs bRSVs hRSV Hrsv oRSV hRSV NS1 8768-69 89 * NS2 92 83-84 87 * N 96 93 60 P — 81 M — 89 F 89 80-81 38-40 G53 88-100 21-29 38-41 60-62 * M2 92 94 41 SH 76 45-50 56 L — *Nodetectable sequence homology

The Genus Metapneumovirus

Avian Pneumoviruses (APV) has been identified as the etiological agentof turkey rhinotracheitis (McDougall, 1986; Collins, 1988) and istherefore often referred to as turkey rhinotracheitis virus (TRTV). Thedisease is an upper respiratory tract infection of turkeys, resulting inhigh morbidity and variable, but often high, mortality. In turkey hens,the virus can also induce substantial reductions in egg production. Thesame virus can also infect chickens, but in this species, the role ofthe virus as a primary pathogen is less clearly defined, although it iscommonly associated with swollen head syndrome (SHS) in breeder chicken(Cook, 2000). The virions are pleiomorphic, though mainly spherical,with sizes ranging from 70 to 600 nm and the nucleocapsid, containingthe linear, non-segmented, negative-sense RNA genome, shows helicalsymmetry (Collins, 1986; Giraud, 1986). This morphology resembles thatof members of the family Paramyxoviridae. Analyses of the APV-encodedproteins and RNAs suggested that of the two subfamilies of this family(Paramyxovirinae and Pneumovirinae), APV most closely resembled thePneumovirinae (Collins, 1988; Ling, 1988; Cavanagh, 1988).

APV has no non-structural proteins (NS1 and NS2) and the gene order(3′-N-P-M-F-M2-SH-G-L-5′) is different from that of mammalianPneumoviruses such as RSV. APV has therefore recently been classified asthe type species for the new genus Metapneumovirus (Pringle, 1999).

Differences in neutralization patterns, ELISA and reactivity withmonoclonal antibodies have revealed the existence of different antigenictypes of APV. Nucleotide sequencing of the G gene led to the definitionof two virus subtypes (A and B), which share only 38% amino acidhomology (Collins, 1993; Juhasz, 1994). An APV isolated from Colorado,USA (Cook, 1999), was shown to cross-neutralize poorly with subtype Aand B viruses and based on sequence information was designated to anovel subtype, C (Seal, 1998; Seal 2000). Two non-A/non-B APVs wereisolated in France, and were shown to be antigenically distinct fromsubtypes A, B and C. Based on amino acid sequences of the F, L and Ggenes, these viruses were classified again as a novel subtype, D(Bayon-Auboyer, 2000).

Diagnosis of APV infection can be achieved by virus isolation in chickenor turkey tracheal organ cultures (TOCs) or in Vero cell cultures. Acytopathic effect (CPE) is generally observed after one or twoadditional passages. This CPE is characterized by scattered focal areasof cell rounding leading to syncytial formation (Buys, 1989). A numberof serology assays, including IF and virus neutralization assays havebeen developed. Detection of antibodies to APV by ELISA is the mostcommonly used method (O'Loan, 1989; Gulati, 2000). Recently, thepolymerase chain reaction (PCR) has been used to diagnose APVinfections. Swabs taken from the esophagus can be used as the startingmaterial (Bayon-Auboyer, 1999; Shin, 2000).

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What is claimed is:
 1. A method for diagnosing a human metapneumovirusinfection in a mammal, the method comprising: determining in a samplefrom the mammal the presence of a component of human metapneumovirus byhybridizing a nucleic acid amplification product of the sample with aDNA molecule of at least 10 nucleotides in length that is at least 90%identical to a stretch of the DNA sequence of SEQ ID NO: 36, or thecomplement thereof, wherein the nucleic acid amplification product isthe product of a nucleic acid amplification reaction with a nucleic acidfrom the sample as the template for the reaction; wherein hybridizationof the DNA molecule with the nucleic acid amplification productindicates human metapneumovirus infection of the mammal.
 2. The methodaccording to claim 1, wherein the nucleic acid amplification reaction isselected from the group consisting of PCR, RAP, and RT-PCR.
 3. Themethod according to claim 1, wherein the nucleic acid amplificationproduct is a DNA molecule.
 4. The method according to claim 1, whereinthe DNA molecule is at least 18 nucleotides in length.
 5. The methodaccording to claim 4, wherein the DNA molecule is at least 90% identicalto a stretch of nucleotides in bases 27-1277 of DNA sequence of SEQ IDNO: 36, or the complement thereof.
 6. The method according to claim 1,wherein the DNA molecule is at least 25 nucleotides in length.
 7. Themethod according to claim 6, wherein the DNA molecule is at least 90%identical to a stretch of nucleotides in bases 27-1277 of DNA sequenceof SEQ ID NO: 36, or the complement thereof.
 8. The method according toclaim 1, wherein the DNA molecule is at least 90% identical to a stretchof nucleotides in bases 27-1277 of DNA sequence of SEQ ID NO: 36, or thecomplement thereof.
 9. The method according to claim 1, wherein the DNAmolecule comprises SEQ ID NO:
 122. 10. A method for diagnosing a humanmetapneumovirus infection of a mammal, the method comprising:determining in a sample from the mammal the presence of humanmetapneumovirus or a component thereof by hybridizing a nucleic acidamplification product of the sample with a DNA molecule specificallyreactive with a stretch of at least 10 nucleotides of the DNA sequenceof SEQ ID NO: 36, or the complement thereof, wherein the nucleic acidamplification product is the product of a nucleic acid amplificationreaction with a nucleic acid from the sample as the template for thereaction; wherein hybridization of the DNA molecule with the nucleicacid amplification product indicates human metapneumovirus infection ofthe mammal.
 11. The method according to claim 10, wherein nucleic acidamplification reaction is selected from the group consisting of PCR,RAP, and RT-PCR.
 12. The method according to claim 10, wherein thenucleic acid amplification product is a DNA molecule.
 13. The methodaccording to claim 10, wherein the DNA molecule is at least 18nucleotides in length.
 14. The method according to claim 10, wherein theDNA is at least 25 nucleotides in length.
 15. The method according toclaim 10, wherein the DNA molecule is specifically reactive with astretch of at least 18 nucleotides of the DNA sequence of SEQ ID NO: 36.16. The method according to claim 10, wherein the DNA molecule isspecifically reactive with a stretch of at least 25 nucleotides of theDNA sequence of SEQ ID NO:
 36. 17. A method of detecting if humanmetapneumovirus is present in a mammalian subject, the methodcomprising: contacting an amplified product with a DNA molecule thathybridizes with the amplified product; hybridizing the DNA molecule tothe amplified product; and detecting the hybridized DNA molecule;wherein the amplified product is the product of a nucleic acidamplification reaction with a set of primers for the amplification of atleast a portion of the N gene of human metapneumovirus and a nucleicacid from the sample as the template for the reaction, wherein the Ngene, when transcribed and translated, produces a polypeptide at least90% identical to SEQ ID NO: 1; and wherein the detection of thehybridized DNA molecule indicates the presence of human metapneumovirusin the mammalian subject.
 18. The method according to claim 17, whereinthe nucleic acid amplification reaction is selected from the groupconsisting of PCR, RAP, and RT-PCR.
 19. The method according to claim17, wherein the DNA molecule comprises a label.
 20. The method accordingto claim 17, wherein detecting the hybridized DNA molecule comprisesdetecting the label.
 21. The method according to claim 17, wherein theDNA molecule is at least 10 bases in length.
 22. The method according toclaim 17, wherein the DNA molecule is at least 40 bases in length. 23.The method according to claim 17, wherein the DNA molecule comprises SEQID NO:
 122. 24. The method according to claim 17, wherein the set ofprimers comprises SEQ ID NOs: 113 and
 114. 25. The method according toclaim 17, wherein the N gene comprises bases 27-1227 of SEQ ID NO:36.26. A method of detecting if human metapneumovirus is present in amammalian subject, the method comprising: contacting an amplifiedproduct with a DNA molecule of at least 25 nucleotides in length thatspecifically hybridizes with the amplified product; hybridizing the DNAmolecule to the amplified product; and detecting the hybridized DNAmolecule; wherein the amplified product is the product of a nucleic acidamplification reaction with a set of primers for the amplification of atleast a portion of the N gene of human metapneumovirus and a nucleicacid from the sample as the template for the reaction, wherein the Ngene, when transcribed and translated, produces a polypeptide at least90% identical to SEQ ID NO: 1; and wherein detection of the hybridizedDNA molecule indicates the presence of human metapneumovirus in themammalian subject.